Anti-TMPRSS2 antibodies and antigen-binding fragments

ABSTRACT

The present invention includes an antibody or antigen-binding fragment thereof that binds specifically to TMPRSS2 and methods of using such antibodies and fragments for treating or preventing viral infections (e.g., influenza virus infections).

This application claims the benefit of U.S. provisional patentapplication No. 62/622,292, filed Jan. 26, 2018; which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies and antigen-bindingfragments that bind specifically to TMPRSS2 and methods for treating orpreventing viral infections with said antibodies and fragments.

BACKGROUND OF THE INVENTION

Influenza viruses have acquired resistance to currently used drugs thattarget the viral neuraminidase (NA) or the ion channel protein, matrixprotein 2 (M2). The emergence of drug resistance highlights the need forthe development of novel antiviral strategies. Host cell targeting mayreduce or avoid the emergence of escape mutants, but could create a“sink” due to widespread expression and raise the concern for toxicity.A number of respiratory virus fusion proteins have been shown to requirecleavage by host protease(s) for activation (Shirato et al. ClinicalIsolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry.Journal of Virology. 91, e01387-16 (2017); Reinke et al., Differentresidues in the SARS-CoV spike protein determine cleavage and activationby the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017); Zhouet al., Protease inhibitors targeting coronavirus and filovirus entry.Antiviral Research. 116, 76-84 (2015); Zmora et al. TMPRSS2 Isoform 1Activates Respiratory Viruses and Is Expressed in Viral Target Cells.PLoS ONE. 10, e0138380 (2015)), including influenza (Zmora et al.,Non-human primate orthologues of TMPRSS2 cleave and activate theinfluenza virus hemagglutinin. PLoS ONE. 12, e0176597 (2017);Böttcher-Friebertshäuser et al., Inhibition of influenza virus infectionin human airway cell cultures by an antisense peptide-conjugatedmorpholino oligomer targeting the hemagglutinin-activating proteaseTMPRSS2. Journal of Virology. 85, 1554-1562 (2011); Bertram et al.,TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenzavirus in Caco-2 cells. Journal of Virology. 84, 10016-10025 (2010);Tarnow et al., TMPRSS2 is a host factor that is essential forpneumotropism and pathogenicity of H7N9 influenza A virus in mice.Journal of Virology (2014), May; 88(9):4744-51).

Influenza A hemagglutinin precursor (HA0) requires cleavage by a hostserine protease, to HA1 and HA2, for activation. For example,transmembrane protease, serine 2; TMPRSS2, TMPRSS4 and TMPRSS11D as wellas human airway trypsin-like protease (HAT) have been implicated in HAcleavage (Bertram et al., TMPRSS2 and TMPRSS4 facilitatetrypsin-independent spread of influenza virus in Caco-2 cells. Journalof Virology. 84, 10016-10025 (2010); Bottcher et al., ProteolyticActivation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT fromHuman Airway Epithelium. Journal of Virology. 2006 October;80(19):9896-8; International patent application publication no.WO2017/151453). Also, TMPRSS2 is a target for anti-cancer therapy. Seee.g., WO2008127347 and WO2002004953. A fusion between TMPRSS2 and ERG(TMPRSS2:ERG) is a gene fusion known to be a major driver of prostatecarcinogenesis which is triggered by the ERα and repressed by the ERβ.Bonkhoff, Estrogen receptor signaling in prostate cancer: Implicationsfor carcinogenesis and tumor progression, Prostate 78(1): 2-10 (2018).

SUMMARY OF THE INVENTION

Although there are small molecule inhibitors of TMPRSS2 and researchantibodies, useful, for example, for immunohistochemistry, there is aneed in the art for neutralizing therapeutic anti-TMPRSS2 antibodies andtheir use for treating or preventing viral infection. See e.g., Shen etal. Biochimie 142: 1-10 (2017), WO2008127347; WO2002004953; U.S. Pat.No. 9,498,529; antibody ab92323, available from Abcam (Cambridge, Mass.)or antibodies sc-515727 and sc-101847 available from Santa Cruz Biotech(Dallas, Tex.). The present invention addresses this need, in part, byproviding human anti-human TMPRSS2 antibodies, such as H1H7017N, andcombinations thereof including, for example, anti-influenza HAantibodies (e.g., Group I HA or Group II HA) and methods of use thereoffor treating viral infections.

The present invention provides a neutralizing human antigen-bindingprotein that specifically binds to human TMPRSS2, for example, anantibody or antigen-binding fragment thereof. For example, in anembodiment of the invention, the antigen-binding protein comprises: (a)the CDR-H1, CDR-H2, and CDR-H3 of an immunoglobulin heavy chain thatcomprises the amino acid sequence set forth in SEQ ID NO: 2, 17 or 19;and/or (b) the CDR-L1, CDR-L2, and CDR-L3 of an immunoglobulin lightchain that comprises the amino acid sequence set forth in SEQ ID NO: 4or 18. In an embodiment of the invention, the antigen-binding proteincomprises: (a) a light chain immunoglobulin variable region comprisingan amino acid sequence having at least 90% amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO: 4 or 18; and/or (b) aheavy chain immunoglobulin variable region comprising an amino acidsequence having at least 90% amino acid sequence identity to the aminoacid sequence set forth in SEQ ID NO: 2, 17 or 19. In an embodiment ofthe invention, the present invention provides antigen-binding proteincomprising: (a) CDR-L1, CDR-L2 and CDR-L3 of a light chainimmunoglobulin comprising an amino acid sequence set forth in SEQ ID NO:4 or 18 and at least 90% amino acid sequence identity to the amino acidsequence set forth in SEQ ID NO: 4 or 18; and/or (b) CDR-H1, CDR-H2 andCDR-H3 of a heavy chain immunoglobulin comprising an amino acid sequenceset forth in SEQ ID NO: 2, 17 or 19 and at least 90% amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 2, 17 or 19.For example, in an embodiment of the invention, the antigen-bindingprotein comprises a light chain immunoglobulin variable region thatcomprises (a) a CDR-H1 comprising the amino acid sequence: G F T F S S YG (SEQ ID NO: 6); (b) a CDR-H2 comprising the amino acid sequence: I W ND G S Y V (SEQ ID NO: 8); (c) a CDR-H3 comprising the amino acidsequence: A R E G E W V L Y Y F D Y (SEQ ID NO: 10); and a heavy chainimmunoglobulin variable region that comprises (a) a CDR-L1 comprisingthe amino acid sequence: Q S I S S W (SEQ ID NO: 12); (b) a CDR-L2comprising the amino acid sequence: K A S (SEQ ID NO: 14); and/or (c) aCDR-L3 comprising the amino acid sequence: Q Q Y N S Y S Y T (SEQ ID NO:16). The present invention also provides an antigen-binding proteincomprising: (a) a heavy chain immunoglobulin that comprises the aminoacid sequence set forth in SEQ ID NO: 17 or 19; and/or (b) a light chainimmunoglobulin that comprises the amino acid sequence set forth in SEQID NO: 18.

The present invention also provides any anti-TMPRSS2 antigen-bindingprotein that competes with any antigen-binding protein that is set forthherein for binding to TMPRSS2 (e.g., as determined by use of using areal time, label-free bio-layer interferometry assay, e.g., on an OctetRED384 biosensor (Pall ForteBio Corp.)); or which binds to the same oran overlapping epitope on TMPRSS2 (or a fragment thereof) as anyantigen-binding protein that is set forth herein.

The present invention also provides multispecific antigen-bindingproteins that bind to TMPRSS2 and another antigen or to TMPRSS2 at adifferent epitope. For example, the multispecific molecule comprises (a)a first antigen-binding domain that binds specifically to TMPRSS2; and(b) a second antigen-binding domain that binds specifically to anotherantigen or to TMPRSS2 or to an epitope which differs from that of thefirst antigen-binding domain.

The present invention also provides any anti-TMPRSS2 antigen-bindingprotein (e.g., an antibody or antigen-binding fragment, e.g., comprisinga sequence set forth herein) that comprises one or more of the followingproperties:

-   -   Inhibits growth of influenza virus (e.g., A/Puerto Rico/08/1934        (H1N1)) in TMPRSS2-expressing cells (e.g., Calu-3 cells);    -   Binds to the surface of TMPRSS-expressing cells (e.g.,        MDCK/Tet-on), e.g., with an EC₅₀ value of 440 pM or 1.06 nM;    -   Does not significantly bind to MDCK/Tet-on cells which do not        express TMPRSS2;    -   Binds to human TMPRSS2 with a K_(D) of about 2.81×10⁻⁹M at about        25° C.;    -   Binds to human TMPRSS2 with a K_(D) of about 9.31×10⁻⁹M at about        37° C.;    -   Binds to cynomolgous TMPRSS2 with a K_(D) of about 5.60×10⁻⁸M at        about 25° C.;    -   Binds to cynomolgous TMPRSS2 with a K_(D) of about 1.40×10⁻⁷M at        about 37° C.;    -   Limits spread of influenza virus infection of cells in vitro;        and/or    -   Protects a mouse engineered to express the human TMPRSS2 protein        from death caused by influenza virus infection.

The present invention also provides a complex comprising anyantigen-binding protein set forth herein bound to a TMPRSS2 polypeptide,e.g., in vitro or in the body of a subject.

The present invention also provides a method for making an anti-TMPRSS2antigen-binding protein set forth herein (e.g., H1H7017N) orimmunoglobulin chain thereof comprising: (a) introducing one or morepolynucleotides encoding a light and/or a heavy immunoglobulin chain ofthe said antigen-binding protein; (b) culturing the host cell (e.g., CHOcell, Pichia cell or Pichia pastoris cell) under conditions favorable toexpression of the polynucleotide; and (c) optionally, isolating theantigen-binding protein or immunoglobulin chain from the host celland/or medium in which the host cell is grown. An antigen-bindingprotein or immunoglobulin chain which is a product of such a method ispart of the present invention.

A polypeptide (e.g., an immunoglobulin) comprising: (a) CDR1, CDR2, andCDR3 of a V_(H) domain of an immunoglobulin chain that comprises theamino acid sequence set forth in SEQ ID NO: 2; or (b) CDR1, CDR2, andCDR3 of a V_(L) domain of an immunoglobulin chain that comprises theamino acid sequence set forth in SEQ ID NO: 4 (e.g., wherein thepolypeptide is in a host cell) also forms part of the present invention.

The present invention also provides a polynucleotide (e.g., DNA or RNA)that encoded a polypeptide of the present invention. In an embodiment ofthe invention, the polynucleotide encodes two different immunoglobulinchains (e.g., heavy and light). In an embodiment of the invention, onepolynucleotide encodes a light immunoglobulin chain and anotherpolynucleotide encodes a heavy immunoglobulin chain, e.g., wherein thechains are in a host cell or are in a vessel. For example, thepolynucleotide is in a vector (e.g., a plasmid) and/or is integratedinto a host cell chromosome.

Host cells (e.g., CHO cell, Pichia cell or Pichia pastoris cell) of thepresent invention may include an anti-TMPRSS2 antigen-binding protein(e.g., H1H7017N), polypeptide thereof or polynucleotide encoding such apolypeptide and/or a vector including such a polynucleotide.

The present invention also provides a composition or kit comprising ananti-TMPRSS2 antigen-binding protein set forth herein (e.g., H1H7017N)in association with a further therapeutic agent (e.g., an anti-viraldrug and/or a vaccine). For example, the composition may be apharmaceutical composition comprising the antigen-binding protein andpharmaceutically acceptable carrier and, optionally, a furthertherapeutic agent. The further therapeutic agent may be ledipasvir,sofosbuvir, a combination of ledipasvir and sofosbuvir, oseltamivir,zanamivir, ribavirin and interferon-alpha2b, interferon-alpha2a and/oran antibody or antigen-binding fragment thereof that specifically bindsto influenza HA. In an embodiment of the invention, the furthertherapeutic agent is an antibody or antigen binding fragment thereofselected from the group consisting of H1H14611N2; H1H14612N2; H1H11723P;H1H11729P; H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N;H1H11830N2; H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H11711P;H1H11714P; H1H11717P; H1H11724P; H1H11727P; H1H11730P2; H1H11731P2;H1H11734P2; H1H11736P2; H1H11742P2; H1H11744P2; H1H11745P2; H1H11747P2;H1H11748P2; H1H17952B; H1H17953B; H1H17954B; H1H17955B; H1H17956B;H1H17957B; H1H17958B; H1H17959B; H1H17960B; H1H17961B; H1H17962B;H1H17963B; H1H17964B; H1H17965B; H1H17966B; H1H17967B; H1H17968B;H1H17969B; H1H17970B; H1H17971B; H1H17972B; H1H17973B; H1H17974B;H1H17975B; H1H17976B; H1H17977B; H1H17978B; H1H17979B; H1H17980B;H1H17981B; H1H17982B; H1H17983B; H1H17984B; H1H17985B; H1H17986B;H1H17987B; H1H17988B; H1H17989B; H1H17990B; H1H17991B; H1H17992B;H1H17993B; H1H17994B; H1H17995B; H1H17996B; H1H17997B; H1H17998B;H1H17999B; H1H18000B; H1H18001B; H1H18002B; H1H18003B; H1H18004B;H1H18005B; H1H18006B; H1H18007B; H1H18008B; H1H18009B; H1H18010B;H1H18011B; H1H18012B; H1H18013B; H1H18014B; H1H18015B; H1H18016B;H1H18017B; H1H18018B; H1H18019B; H1H18020B; H1H18021B; H1H18022B;H1H18023B; H1H18024B; H1H18025B; H1H18026B; H1H18027B; H1H18028B;H1H18029B; H1H18030B; H1H18031B; H1H18032B; H1H18033B; H1H18034B;H1H18035B; H1H18037B; H1H18038B; H1H18039B; H1H18040B; H1H18041B;H1H18042B; H1H18043B; H1H18044B; H1H18045B; H1H18046B; H1H18047B;H1H18048B; H1H18049B; H1H18051B; H1H18052B; H1H18053B; H1H18054B;H1H18055B; H1H18056B; H1H18057B; H1H18058B; H1H18059B; H1H18060B;H1H18061B; H1H18062B; H1H18063B; H1H18064B; H1H18065B; H1H18066B;H1H18067B; H1H18068B; H1H18069B; H1H18070B; H1H18071B; H1H18072B;H1H18073B; H1H18074B; H1H18075B; H1H18076B; H1H18077B; H1H18078B;H1H18079B; H1H18080B; H1H18081B; H1H18082B; H1H18083B; H1H18084B;H1H18085B; H1H18086B; H1H18087B; H1H18088B; H1H18089B; H1H18090B;H1H18091B; H1H18092B; H1H18093B; H1H18094B; H1H18095B; H1H18096B;H1H18097B; H1H18098B; H1H18099B; H1H18100B; H1H18101B; H1H18102B;H1H18103B; H1H18104B; H1H18105B; H1H18107B; H1H18108B; H1H18109B;H1H18110B; H1H18111B; H1H18112B; H1H18113B; H1H18114B; H1H18115B;H1H18116B; H1H18117B; H1H18118B; H1H18119B; H1H18120B; H1H18121B;H1H18122B; H1H18123B; H1H18124B; H1H18125B; H1H18126B; H1H18127B;H1H18128B; H1H18129B; H1H18130B; H1H18131B; H1H18132B; H1H18133B;H1H18134B; H1H18135B; H1H18136B; H1H18137B; H1H18138B; H1H18139B;H1H18140B; H1H18141B; H1H18142B; H1H18143B; H1H18144B; H1H18145B;H1H18146B; H1H18147B; H1H18148B; H1H18149B; H1H18150B; H1H18151B;H1H18152B; H1H18153B; H1H18154B; H1H18155B; H1H18156B; H1H18157B;H1H18158B; H1H18159B; H1H18160B; H1H18161B; H1H18162B; H1H18163B;H1H18164B; H1H18165B; H1H18166B; H1H18167B; H1H18168B; H1H18169B;H1H18170B; H1H18171B; H1H18172B; H1H18173B; H1H18174B; H1H18175B;H1H18176B; H1H18177B; H1H18178B; H1H18179B; H1H18180B; H1H18181B;H1H18182B; H1H18183B; H1H18184B; H1H18185B; H1H18186B; H1H18187B;H1H18188B; H1H18189B; H1H18190B; H1H18191B; H1H18192B; H1H18193B;H1H18194B; H1H18195B; H1H18196B; H1H18197B; H1H18198B; H1H18199B;H1H18200B; H1H18201B; H1H18202B; H1H18203B; H1H18204B; H1H18205B;H1H18206B; H1H18207B; H1H18208B; H1H18209B; H1H18210B; H1H18211B;H1H18212B; H1H18213B; H1H18214B; H1H18216B; H1H18217B; H1H18218B;H1H18219B; H1H18220B; H1H18221B; H1H18222B; H1H18223B; H1H18224B;H1H18225B; H1H18226B; H1H18227B; H1H18228B; H1H18229B; H1H18230B;H1H18231B; H1H18232B; H1H18233B; H1H18234B; H1H18235B; H1H18236B;H1H18237B; H1H18238B; H1H18239B; H1H18240B; H1H18241B; H1H18242B;H1H18243B; H1H18244B; H1H18245B; H1H18246B; H1H18247B; H1H18248B;H1H18249B; H1H18250B; H1H18251B; H1H18252B; H1H18253B; H1H18254B;H1H18255B; H1H18256B; H1H18257B; H1H18258B; H1H18259B; H1H18261B;H1H18262B; H1H18263B; H1H18264B; H1H18265B; H1H18266B; H1H18267B;H1H18268B; H1H18269B; H1H18270B; H1H18271B; H1H18272B; H1H18274B;H1H18275B; H1H18276B; H1H18277B; H1H18278B; H1H18279B; H1H18280B;H1H18281B; H1H18282B; H1H18283B; H1H18284B; H1H18285B; H1H18286B;H1H18287B; H1H18288B; H1H18289B; H1H18290B; H1H18291B; H1H18292B;H1H18293B; H1H18294B; H1H18295B; H1H18297B; H1H18298B; H1H18299B;H1H18300B; H1H18301B; H1H18302B; H1H18303B; H1H18304B; H1H18305B;H1H18306B; H1H18307B; H1H18308B; H1H18309B; H1H18310B; H1H18311B;H1H18312B; H1H18313B; H1H18314B; H1H18315B; H1H18316B; H1H18317B;H1H18318B; H1H18319B; H1H18320B; H1H18321B; H1H18322B; H1H18323B;H1H18324B; H1H18325B; H1H18326B; H1H18327B; H1H18328B; H1H18329B;H1H18330B; H1H18331B; H1H18332B; H1H18333B; H1H18334B; and H1H18335B.

In an embodiment of the invention, a further therapeutic agent which isprovided in association with an anti-TMPRSS2 antigen-binding protein isan antibody or antigen-binding fragment that binds to influenza Group IIHA protein, such as H1H14611N2; or an antibody or fragment thatcomprises V_(H) and V_(L) of H1H14611N2; or a heavy chain immunoglobulincomprising CDR-H1, CDR-H2 and CDR-H3 of H1H14611N2 (e.g., SEQ ID NOs:25-27) and a light chain immunoglobulin comprising CDR-L1, CDR-L2 andCDR-L3 of H1H14611N2 (e.g., SEQ ID NOs: 29-31).

In an embodiment of the invention, a further therapeutic agent which isprovided in association with an anti-TMPRSS2 antigen-binding protein isan antibody or antigen-binding fragment that binds to influenza Group IIHA protein, such as H1H14612N2; or an antibody or fragment thatcomprises V_(H) and V_(L) of H1H14612N2; or a heavy chain immunoglobulincomprising CDR-H1, CDR-H2 and CDR-H3 of H1H14612N2 (e.g., SEQ ID NOs:41-43) and a light chain immunoglobulin comprising CDR-L1, CDR-L2 andCDR-L3 of H1H14612N2 (e.g., SEQ ID NOs: 45-47).

In an embodiment of the invention, a further therapeutic agent which isprovided in association with an anti-TMPRSS2 antigen-binding protein isan antibody or antigen-binding fragment that binds to influenza Group IHA protein, such as H1H11729P; or an antibody or fragment that comprisesV_(H) and V_(L) of H1H11729P; or a heavy chain immunoglobulin comprisingCDR-H1, CDR-H2 and CDR-H3 of H1H11729P (e.g., SEQ ID NOs: 33-35) and alight chain immunoglobulin comprising CDR-L1, CDR-L2 and CDR-L3 ofH1H11729P (e.g., SEQ ID NOs: 37-39).

The present invention also provides a vessel or injection device thatcomprises an anti-TMPRSS2 antigen-binding protein (e.g., H1H7017N) orcomposition thereof (e.g., pharmaceutical composition).

The present invention also provides a method for treating or preventinga viral infection other than an influenza virus infection, in a subject(e.g., a human) in need thereof, comprising administering atherapeutically effective amount of anti-TMPRSS2 antigen-binding proteinset forth herein (e.g., H1H7017N).

The present invention also provides a method for treating or preventingcancer (e.g., prostate cancer) or infection, e.g., a viral infection,e.g., an infection with an influenza virus, coronavirus, SARS-Co virus,MERS-Co virus, parainfluenza virus, human metapneumovirus or hepatitis Cvirus (HCV), in a subject (e.g., a human) in need thereof, comprisingadministering a therapeutically effective amount of anti-TMPRSS2antigen-binding protein set forth herein (e.g., H1H7017N). For example,the antigen-binding protein is administered in association with one ormore further therapeutic agents (e.g., anti-viral drug and/or avaccine). In an embodiment of the invention, a further therapeutic agentis a member selected from the group consisting of: ledipasvir,sofosbuvir, a combination of ledipasvir and sofosbuvir, oseltamivir,zanamivir, ribavirin and interferon-alpha2b, interferon-alpha2a and anantibody or antigen-binding fragment thereof that specifically binds toinfluenza HA. In an embodiment of the invention, a further therapeuticagent is an antibody or antigen binding fragment thereof selected fromthe group consisting of H1H14611N2; H1H14612N2; H1H11723P; H1H11729P;H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N; H1H11830N2;H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H11711P; H1H11714P;H1H11717P; H1H11724P; H1H11727P; H1H11730P2; H1H11731P2; H1H11734P2;H1H11736P2; H1H11742P2; H1H11744P2; H1H11745P2; H1H11747P2; H1H11748P2;H1H17952B; H1H17953B; H1H17954B; H1H17955B; H1H17956B; H1H17957B;H1H17958B; H1H17959B; H1H17960B; H1H17961B; H1H17962B; H1H17963B;H1H17964B; H1H17965B; H1H17966B; H1H17967B; H1H17968B; H1H17969B;H1H17970B; H1H17971B; H1H17972B; H1H17973B; H1H17974B; H1H17975B;H1H17976B; H1H17977B; H1H17978B; H1H17979B; H1H17980B; H1H17981B;H1H17982B; H1H17983B; H1H17984B; H1H17985B; H1H17986B; H1H17987B;H1H17988B; H1H17989B; H1H17990B; H1H17991B; H1H17992B; H1H17993B;H1H17994B; H1H17995B; H1H17996B; H1H17997B; H1H17998B; H1H17999B;H1H18000B; H1H18001B; H1H18002B; H1H18003B; H1H18004B; H1H18005B;H1H18006B; H1H18007B; H1H18008B; H1H18009B; H1H18010B; H1H18011B;H1H18012B; H1H18013B; H1H18014B; H1H18015B; H1H18016B; H1H18017B;H1H18018B; H1H18019B; H1H18020B; H1H18021B; H1H18022B; H1H18023B;H1H18024B; H1H18025B; H1H18026B; H1H18027B; H1H18028B; H1H18029B;H1H18030B; H1H18031B; H1H18032B; H1H18033B; H1H18034B; H1H18035B;H1H18037B; H1H18038B; H1H18039B; H1H18040B; H1H18041B; H1H18042B;H1H18043B; H1H18044B; H1H18045B; H1H18046B; H1H18047B; H1H18048B;H1H18049B; H1H18051B; H1H18052B; H1H18053B; H1H18054B; H1H18055B;H1H18056B; H1H18057B; H1H18058B; H1H18059B; H1H18060B; H1H18061B;H1H18062B; H1H18063B; H1H18064B; H1H18065B; H1H18066B; H1H18067B;H1H18068B; H1H18069B; H1H18070B; H1H18071B; H1H18072B; H1H18073B;H1H18074B; H1H18075B; H1H18076B; H1H18077B; H1H18078B; H1H18079B;H1H18080B; H1H18081B; H1H18082B; H1H18083B; H1H18084B; H1H18085B;H1H18086B; H1H18087B; H1H18088B; H1H18089B; H1H18090B; H1H18091B;H1H18092B; H1H18093B; H1H18094B; H1H18095B; H1H18096B; H1H18097B;H1H18098B; H1H18099B; H1H18100B; H1H18101B; H1H18102B; H1H18103B;H1H18104B; H1H18105B; H1H18107B; H1H18108B; H1H18109B; H1H18110B;H1H18111B; H1H18112B; H1H18113B; H1H18114B; H1H18115B; H1H18116B;H1H18117B; H1H18118B; H1H18119B; H1H18120B; H1H18121B; H1H18122B;H1H18123B; H1H18124B; H1H18125B; H1H18126B; H1H18127B; H1H18128B;H1H18129B; H1H18130B; H1H18131B; H1H18132B; H1H18133B; H1H18134B;H1H18135B; H1H18136B; H1H18137B; H1H18138B; H1H18139B; H1H18140B;H1H18141B; H1H18142B; H1H18143B; H1H18144B; H1H18145B; H1H18146B;H1H18147B; H1H18148B; H1H18149B; H1H18150B; H1H18151B; H1H18152B;H1H18153B; H1H18154B; H1H18155B; H1H18156B; H1H18157B; H1H18158B;H1H18159B; H1H18160B; H1H18161B; H1H18162B; H1H18163B; H1H18164B;H1H18165B; H1H18166B; H1H18167B; H1H18168B; H1H18169B; H1H18170B;H1H18171B; H1H18172B; H1H18173B; H1H18174B; H1H18175B; H1H18176B;H1H18177B; H1H18178B; H1H18179B; H1H18180B; H1H18181B; H1H18182B;H1H18183B; H1H18184B; H1H18185B; H1H18186B; H1H18187B; H1H18188B;H1H18189B; H1H18190B; H1H18191B; H1H18192B; H1H18193B; H1H18194B;H1H18195B; H1H18196B; H1H18197B; H1H18198B; H1H18199B; H1H18200B;H1H18201B; H1H18202B; H1H18203B; H1H18204B; H1H18205B; H1H18206B;H1H18207B; H1H18208B; H1H18209B; H1H18210B; H1H18211B; H1H18212B;H1H18213B; H1H18214B; H1H18216B; H1H18217B; H1H18218B; H1H18219B;H1H18220B; H1H18221B; H1H18222B; H1H18223B; H1H18224B; H1H18225B;H1H18226B; H1H18227B; H1H18228B; H1H18229B; H1H18230B; H1H18231B;H1H18232B; H1H18233B; H1H18234B; H1H18235B; H1H18236B; H1H18237B;H1H18238B; H1H18239B; H1H18240B; H1H18241B; H1H18242B; H1H18243B;H1H18244B; H1H18245B; H1H18246B; H1H18247B; H1H18248B; H1H18249B;H1H18250B; H1H18251B; H1H18252B; H1H18253B; H1H18254B; H1H18255B;H1H18256B; H1H18257B; H1H18258B; H1H18259B; H1H18261B; H1H18262B;H1H18263B; H1H18264B; H1H18265B; H1H18266B; H1H18267B; H1H18268B;H1H18269B; H1H18270B; H1H18271B; H1H18272B; H1H18274B; H1H18275B;H1H18276B; H1H18277B; H1H18278B; H1H18279B; H1H18280B; H1H18281B;H1H18282B; H1H18283B; H1H18284B; H1H18285B; H1H18286B; H1H18287B;H1H18288B; H1H18289B; H1H18290B; H1H18291B; H1H18292B; H1H18293B;H1H18294B; H1H18295B; H1H18297B; H1H18298B; H1H18299B; H1H18300B;H1H18301B; H1H18302B; H1H18303B; H1H18304B; H1H18305B; H1H18306B;H1H18307B; H1H18308B; H1H18309B; H1H18310B; H1H18311B; H1H18312B;H1H18313B; H1H18314B; H1H18315B; H1H18316B; H1H18317B; H1H18318B;H1H18319B; H1H18320B; H1H18321B; H1H18322B; H1H18323B; H1H18324B;H1H18325B; H1H18326B; H1H18327B; H1H18328B; H1H18329B; H1H18330B;H1H18331B; H1H18332B; H1H18333B; H1H18334B; and H1H18335B.

The present invention also provides a method for administering ananti-TMRPSS2 antigen-binding protein (e.g., H1H7017N) set forth hereininto the body of a subject (e.g., a human) comprising injecting theantigen-binding protein into the body of the subject parenterally (e.g.,subcutaneously, intravenously or intramuscularly).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the progression of the A/Puerto Rico/08/1934 (H1N1)-GFPvirus spreading in different cell lines with an initial multiplicity ofinfection of 0.01 in absence of exogenous trypsin. Calu3 (circle), A549(square), MDCK (triangle) and HepG2 (inverted triangle) cells.

FIG. 1B shows the progression of the A/Puerto Rico/08/1934 (H1N1)-GFPvirus spreading in different cell lines with an initial multiplicity ofinfection of 0.001 in absence of exogenous trypsin. Calu3 (circle), A549(square), MDCK (triangle) and HepG2 (inverted triangle) cells.

FIG. 2. shows application of H1H7017N during the infection cycledecreases the number of Fluorescent Focus Units (FFU) of A/PuertoRico/08/1934 (H1N1) at 72 hours post-infection compared to isotypecontrol antibody, no antibody, anti-HA antibody and uninfected controls.

FIG. 3A shows anti-TMPRSS2, H1H7017N, binds to human and cynomolgousmonkey TMPRSS2 expressed on cells. H1H7017N, bound toMDCK/Tet-on/hTMPRSS2 and MDCK/Tet-on/mfTMPRSS2 with EC50 values of 460pM and 1.06 nM respectively and did not show significant binding toMDCK/Tet-on cells.

FIG. 3B shows anti-TMPRSS2, H1H7017N, binds to human and cynomolgousmonkey TMPRSS2 expressed on cells. Control mAb1, an irrelevant isotypecontrol antibody, did not show binding to any of the cell lines tested.

FIG. 4. shows a survival curve of a mouse engineered to express thehuman TMPRSS2 protein treated with 5 mg/kg of H1H7017N on day −1 PI(inverted triangle, dashed line) or day 0 PI (circle, solid line)showing protection against H1N1 in a prophylactic model. Mice treatedwith the isotype control H1H1238N (triangle, solid line) showed noprotection.

FIG. 5 shows a survival curve of a mouse engineered to express the humanTMPRSS2 protein infected with H1N1, treated with 10 mg/kg H1H7017Ndemonstrating protection. Mice were treated on day 0 (diamond, dottedline), day 1 (circle, solid line), day 2 (inverted triangle, solidline), or day 3 PI (square, dashed line). The isotype control H1H1238N(triangle, solid line) had partial protection with a 25% survival rate.

FIG. 6 shows a survival curve of hTPMRSS2 mice treated with 10 mg/kg ofH1H7017N on day 1 PI (triangle) or day 2 PI (circle) showing protectionagainst H3N2. Untreated mice (square) showed no protection.

FIG. 7A shows a survival curve of wild-type mice infected with 150 PFUs(triangle), 750 PFUs (square), or 1,500 PFUs (circle) of A/PuertoRico/08/1934 (H1N1). Mice were weighed daily until day 14 PI.

FIG. 7B shows a survival curve of mice engineered to express the humanTMPRSS2 protein infected with 150 PFUs (triangle), 750 PFUs (square), or1,500 PFUs (circle) of A/Puerto Rico/08/1934 (H1N1). Mice were weigheddaily until day 14 PI.

FIG. 8 shows a survival curve of a mouse engineered to express the humanTMPRSS2 protein infected with A/Aichi/2/68 (HA, NA)×A/PR/8/34 (H3N2) onday 0 and treated with a combination of 2.5 mg/kg each of H1H7017N andH1H14611N2 (diamond), 10 mg/kg H1H7017N (triangle), 10 mg/kg H1H14611N2(square), 5 mg/kg each of H1H7017N and H1H14611N2, or 10 mg/kg hIgG1isotype control (circle). Mice were weighed daily until day 14 PI.

FIG. 9 shows a survival curve of a mouse engineered to express the humanTMPRSS2 protein infected with A/Puerto Rico/08/1934 (H1N1) on day 1 PIand treated with a combination of 1 mg/kg of H1H7017N and 2 mg/kg ofH1H11729P (circle), 2.5 mg/kg each of H1H7017N and H1H11729P (invertedtriangle), 5 mg/kg H1H11729P (diamond), 5 mg/kg H1H7017N (square), or 5mg/kg hIgG1 isotype control (triangle). Mice were weighed daily untilday 14 PI.

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

The term “influenza hemagglutinin”, also called “influenza HA” is atrimeric glycoprotein found on the surface of influenza virions, whichmediates viral attachment (via HA1 binding to α-2,3- and α-2,6-sialicacids) and entry (through conformational change) into host cells. The HAis comprised of two structural domains: a globular head domaincontaining the receptor binding site (subject to high frequency ofantigenic mutations) and the stem region (more conserved among variousstrains of influenza virus). The influenza HA is synthesized as aprecursor (HA0) that undergoes proteolytic processing to produce twosubunits (HA1 and HA2) which associate with one another to form thestem/globular head structure. The viral HA is the most variable antigenon the virus and the stem (HA2) is highly conserved within each group.

The term “influenza neuraminidase”, also called “influenza NA” is anexosialidase (EC 3.2.1.18) which cleaves α-ketosidic linkage between thesialic (N-acetylneuraminic) acid and an adjacent sugar residue.

The amino acid sequence of full-length Influenza HA is exemplified bythe amino acid sequence of influenza isolate H1N1 A/California/04/2009provided in GenBank as accession number FJ966082.1. The term“influenza-HA” also includes protein variants of influenza HA isolatedfrom different influenza isolates, e.g., GQ149237.1, NC_002017,KM972981.1, etc. The term “influenza-HA” also includes recombinantinfluenza HA or a fragment thereof. The term also encompasses influenzaHA or a fragment thereof coupled to, for example, histidine tag, mouseor human Fc, or a signal sequence.

An anti-TMPRSS2 “antigen-binding protein” is a polypeptide or complex ofmore than one polypeptide (e.g., a tetrameric IgG antibody) that bindsspecifically to TMPRSS2 polypeptide, for example, an anti-TMPRSS2antibody or antigen-binding fragment whether monospecific ormultispecific.

TMPRSS2

TMPRSS2 (Transmembrane protease serine 2) is a protein, located on humanchromosome 21, that belongs to the serine protease family (type IItransmembrane serine proteases (TTSPs)) which is important for influenzavirus infectivity. TMPRSS2 has been demonstrated to mediate cleavage ofinfluenza virus HA0 to HA1 and HA2.

The human TMPRSS2 gene encodes a predicted protein of 492 amino acidswhich anchors to the plasma membrane. The protein converts to its matureform through autocatalytic cleavage between Arg255 and Ile256. Aftercleavage, the mature proteases are mostly membrane bound, yet a portionof them may be liberated into the extracellular milieu.

In an embodiment of the invention, human TMPRSS2 (V160M) comprises theamino acid sequence:

(SEQ ID NO: 22; methionine 160 in bold font)MALNSGSPPAIGPYYENHGYQPENPYPAQPTVVPTVYEVHPAQYYPSPVPQYAPRVLTQASNPVVCTQPKSPSGTVCTSKTKKALCITLTLGTFLVGAALAAGLLWKFMGSKCSNSGIECDSSGTCINPSNWCDGVSHCPGGEDENRCVRLYGPNFILQMYSSQRKSWHPVCQDDWNENYGRAACRDMGYKNNFYSSQGIVDDSGSTSFMKLNTSAGNVDIYKKLYHSDACSSKAVVSLRCIACGVNLNSSRQSRIVGGESALPGAWPWQVSLHVQNVHVCGGSIITPEWIVTAAHCVEKPLNNPWHWTAFAGILRQSFMFYGAGYQVEKVISHPNYDSKTKNNDIALMKLQKPLTFNDLVKPVCLPNPGMMLQPEQLCWISGWGATEEKGKTSEVLNAAKVLLIETQRCNSRYVYDNLITPAMICAGFLQGNVDSCQGDSGGPLVTSKNNIWWLIGDTSWGSGCAKAYRPGVYGNVMVFTDWIYRQMRADG.In an embodiment of the invention, the TMPRSS2 polypeptide does notcomprise the V160M mutation. See also NM_005656.3.

In an embodiment of the invention, Macaca mulatta TMPRSS2 (S129L, N251S,I415V, R431Q, D492G) comprises the amino acid sequence:

(SEQ ID NO: 23) MALNSGSPPGVGPYYENHGYQPENPYPAQPTVAPNVYEVHPAQYYPSPVPQYTPRVLTHASNPAVCRQPKSPSGTVCTSKTKKALCVTMTLGAVLVGAALAAGLLWKFMGSKCSDSGIECDSSGTCISLSNWCDGVSHCPNGEDENRCVRLYGPNFILQVYSSQRKSWHPVCRDDWNENYARAACRDMGYKNSFYSSQGIVDNSGATSFMKLNTSAGNVDIYKKLYHSDACSSKAVVSLRCIACGVRSNLSRQSRIVGGQNALLGAWPWQVSLHVQNIHVCGGSIITPEWIVTAAHCVEKPLNSPWQWTAFVGTLRQSSMFYEKGHRVEKVISHPNYDSKTKNNDIALMKLHTPLTFNEVVKPVCLPNPGMMLEPEQHCWISGWGATQEKGKTSDVLNAAMVPLIEPRRCNNKYVYDGLITPAMICAGFLQGTVDSCQGDSGGPLVTLKNDVWWLIGDTSWGSGCAQANRPGVYGNVTVFTDWIYRQMRADG.In an embodiment of the invention, the TMPRSS2 polypeptide does notcomprise the S129L, N251S, I415V, R431Q and/or D492G mutation.

In an embodiment of the invention, Mus musculus TMPRSS2 mRNA comprisesthe nucleotide sequence set forth in NM_015775.2.

Viruses

The present invention includes methods for treating or preventing aviral infection in a subject. The term “virus” includes any virus whoseinfection in the body of a subject is treatable or preventable byadministration of an anti-TMPRSS2 antibody or antigen-binding fragmentthereof (e.g., wherein infectivity of the virus is at least partiallydependent on TMPRSS2). In an embodiment of the invention, a “virus” isany virus that expresses HA0 or another substrate of TMPRSS2 whoseproteolytic cleavage is required for full infectivity of the virusagainst a cell in a host. The term “virus” also includes aTMPRSS2-dependent respiratory virus which is a virus that infects therespiratory tissue of a subject (e.g., upper and/or lower respiratorytract, trachea, bronchi, lungs) and is treatable or preventable byadministration of an anti-TMPRSS2. For example, in an embodiment of theinvention, virus includes influenza virus, coronavirus, SARS-Co virus(severe acute respiratory syndrome coronavirus), MERS-Co virus (middleeast respiratory syndrome (MERS) CoV), parainfluenza virus, sendai virus(SeV), human metapneumovirus and/or hepatitis C virus (HCV). “Viralinfection” refers to the invasion and multiplication of a virus in thebody of a subject. The present invention includes embodiments with aproviso that “virus” excludes influenza virus, e.g., wherein viralinfection excludes influenza virus infection.

There are now two genera of human parainfluenza virus (HPIV),respirovirus (HPIV-1 and HPIV-3) and rubulavirus (HPIV-2 and HPIV-4).Both genera (paramyxoviruses) can be separated morphologically frominfluenza virus.

Sendai virus, also known as murine parainfluenza virus, is the typespecies in the genus respirovirus, which also contains the species humanparainfluenza virus 3, bovine parainfluenza virus 3, and humanparainfluenza virus 1. TMPRSS2 Is an Activating Protease for RespiratoryParainfluenza Viruses such as parainfluenza viruses and Sendai virus(SeV). See et al. Abe et al., J. Virol. 87(21): 11930-11935 (2013).

Human metapneumovirus (HMPV) is classified as the first human member ofthe Metapneumovirus genus in the Pneumovirinae subfamily within theParamyxoviridae family. It is an enveloped negative-sensesingle-stranded RNA virus. The RNA genome includes 8 genes coding for 9different proteins. HMPV is identical in gene order to the avianpneumovirus (AMPV), which also belongs to the Metapneumovirus genus.TMPRSS2 is expressed in the human lung epithelium, cleaves the HMPV Fprotein efficiently and supports HMPV multiplication and may be involvedin the development of lower respiratory tract illness in HMPV-infectedpatients. See et al. Shirogane et al. J Virol. 82(17): 8942-8946 (2008).

Hepatitis C virus (HCV) is a small, enveloped, positive-sensesingle-stranded RNA virus of the family Flaviviridae. HCV, with at least6 genotypes and numerous subtypes, is a member of the hepacivirus genus.TMPRSS2 may activate HCV infection at the post-binding and entry stage.Esumi et al., Hepatology 61(2): 437-446 (2015).

Influenza viruses are members of the family Orthomyxoviridae. Thisfamily represents enveloped viruses the genome of which has segmentednegative-sense single-strand RNA segments. There are four genera of thisfamily: types A, B, C and Thogotovirus. The Influenza viruses classes,A, B and C, are based on core protein and are further divided intosubtypes determined by the viral envelope glycoproteins hemagglutinin(HA) and neuraminidase (NA) (e.g., subtype A/H1N1). There are at least18 influenza hemagglutinin (“HA”) protein subtypes (H1-H18 or HA1-HA18)and at least 11 influenza neuraminidase (NA) protein subtypes (N1-N11 orNA1-NA11) used to define influenza subtypes. Group 1 influenza has H1,H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18 subtypes and NA8,NA5, Na4 and NA1 subtypes. Group 2 has H3, H4, H7, H10, H14 and H15subtypes and NA6, NA9, NA7, NA2 and NA3 subtypes. Influenza A virusesinfect a range of mammalian and avian species, whereas type B and Cinfections are largely restricted to humans. The eight genome segmentsof influenza A and B viruses are loosely encapsidated by thenucleoprotein.

Coronavirus virions are spherical with diameters of approximately 125nm. The most prominent feature of coronaviruses is the club-shape spikeprojections emanating from the surface of the virion. These spikes are adefining feature of the virion and give them the appearance of a solarcorona, prompting the name, coronaviruses. Within the envelope of thevirion is the nucleocapsid. Coronaviruses have helically symmetricalnucleocapsids, which is uncommon among positive-sense RNA viruses, butfar more common for negative-sense RNA viruses. Both MERS-CoV (middleeast respiratory syndrome coronavirus) and SARS-CoV (severe acuterespiratory syndrome coronavirus) belong to the coronavirus family. Theinitial attachment of the virion to the host cell is initiated byinteractions between the S protein and its receptor. The sites ofreceptor binding domains (RBD) within the 51 region of a coronavirus Sprotein vary depending on the virus, with some having the RBD at theC-terminus of 51. The 5-protein/receptor interaction is the primarydeterminant for a coronavirus to infect a host species and also governsthe tissue tropism of the virus. Many coronaviruses utilize peptidasesas their cellular receptor. Following receptor binding, the virus mustnext gain access to the host cell cytosol. This is generallyaccomplished by acid-dependent proteolytic cleavage of S protein by acathepsin, TMPRRS2 or another protease, followed by fusion of the viraland cellular membranes.

Anti-TMPRSS2 Antibodies and Antigen-Binding Fragments

The present invention provides antigen-binding proteins, such asantibodies and antigen-binding fragments thereof, that specifically bindto TMPRSS2 protein or an antigenic fragment thereof.

The term “antibody”, as used herein, refers to immunoglobulin moleculescomprising four polypeptide chains, two heavy chains (HCs) and two lightchains (LCs) inter-connected by disulfide bonds (i.e., “full antibodymolecules”), as well as multimers thereof (e.g. IgM)—for example,H1H7017N. Each heavy chain comprises a heavy chain variable region(“HCVR” or “V_(H)”) (e.g., SEQ ID NO 2) and a heavy chain constantregion (comprised of domains C_(H)1, C_(H)2 and C_(H)3). Each lightchain is comprised of a light chain variable region (“LCVR or “V_(L)”)(e.g., SEQ ID NO 4) and a light chain constant region (C_(L)). The V_(H)and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) comprises three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of theinvention, the FRs of the antibody (or antigen binding fragment thereof)are identical to the human germline sequences, or are naturally orartificially modified.

Typically, the variable domains of both the heavy and lightimmunoglobulin chains comprise three hypervariable regions, also calledcomplementarity determining regions (CDRs), located within relativelyconserved framework regions (FR). In general, from N-terminal toC-terminal, both light and heavy chains variable domains comprise FR1,CDR1, FR2, CDR2, FR3, CDR3 and FR4. In an embodiment of the invention,the assignment of amino acids to each domain is in accordance with thedefinitions of Sequences of Proteins of Immunological Interest, Kabat,et al.; National Institutes of Health, Bethesda, Md.; 5^(th) ed.; NIHPubl. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat,et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) JMol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

The present invention includes monoclonal anti-TMPRSS2 antigen-bindingproteins, e.g., antibodies and antigen-binding fragments thereof, aswell as monoclonal compositions comprising a plurality of isolatedmonoclonal antigen-binding proteins. The term “monoclonal antibody”, asused herein, refers to a population of substantially homogeneousantibodies, i.e., the antibody molecules comprising the population areidentical in amino acid sequence except for possible naturally occurringmutations that may be present in minor amounts. A “plurality” of suchmonoclonal antibodies and fragments in a composition refers to aconcentration of identical (i.e., as discussed above, in amino acidsequence except for possible naturally occurring mutations that may bepresent in minor amounts) antibodies and fragments which is above thatwhich would normally occur in nature, e.g., in the blood of a hostorganism such as a mouse or a human.

In an embodiment of the invention, an anti-TMPRSS2 antigen-bindingprotein, e.g., antibody or antigen-binding fragment comprises a heavychain constant domain, e.g., of the type IgA (e.g., IgA1 or IgA2), IgD,IgE, IgG (e.g., IgG1, IgG2, IgG3 and IgG4) or IgM. In an embodiment ofthe invention, an antigen-binding protein, e.g., antibody orantigen-binding fragment comprises a light chain constant domain, e.g.,of the type kappa or lambda.

The term “human” antigen-binding protein, such as an antibody, as usedherein, includes antibodies having variable and constant regions derivedfrom human germline immunoglobulin sequences whether in a human cell orgrafted into a non-human cell, e.g., a mouse cell. See e.g., U.S. Pat.No. 8,502,018, 6,596,541 or 5,789,215. The human mAbs of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include mAbs in which CDRsequences derived from the germline of another mammalian species (e.g.,mouse) have been grafted onto human FR sequences. The term includesantibodies recombinantly produced in a non-human mammal or in cells of anon-human mammal. The term is not intended to include antibodiesisolated from or generated in a human subject. See below.

The present invention includes anti-TMPRSS2 chimeric antigen-bindingproteins, e.g., antibodies and antigen-binding fragments thereof, andmethods of use thereof. As used herein, a “chimeric antibody” is anantibody having the variable domain from a first antibody and theconstant domain from a second antibody, where the first and secondantibodies are from different species. (U.S. Pat. No. 4,816,567; andMorrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855).

The term “recombinant” antigen-binding proteins, such as antibodies orantigen-binding fragments thereof, refers to such molecules created,expressed, isolated or obtained by technologies or methods known in theart as recombinant DNA technology which include, e.g., DNA splicing andtransgenic expression. The term includes antibodies expressed in anon-human mammal (including transgenic non-human mammals, e.g.,transgenic mice), or a cell (e.g., CHO cells) expression system orisolated from a recombinant combinatorial human antibody library.

Recombinant anti-TMPRSS2 antigen-binding proteins, e.g., antibodies andantigen-binding fragments, disclosed herein may also be produced in anE. coli/T7 expression system. In this embodiment, nucleic acids encodingthe anti-TMPRSS2 antibody immunoglobulin molecules of the invention(e.g., H1H7017N) may be inserted into a pET-based plasmid and expressedin the E. coli/T7 system. For example, the present invention includesmethods for expressing an antibody or antigen-binding fragment thereofor immunoglobulin chain thereof in a host cell (e.g., bacterial hostcell such as E. coli such as BL21 or BL21DE3) comprising expressing T7RNA polymerase in the cell which also includes a polynucleotide encodingan immunoglobulin chain that is operably linked to a T7 promoter. Forexample, in an embodiment of the invention, a bacterial host cell, suchas an E. coli, includes a polynucleotide encoding the T7 RNA polymerasegene operably linked to a lac promoter and expression of the polymeraseand the chain is induced by incubation of the host cell with IPTG(isopropyl-beta-D-thiogalactopyranoside). See U.S. Pat. Nos. 4,952,496and 5,693,489 or Studier & Moffatt, Use of bacteriophage T7 RNApolymerase to direct selective high-level expression of cloned genes, J.Mol. Biol. 1986 May 5; 189(1): 113-30.

There are several methods by which to produce recombinant antibodieswhich are known in the art. One example of a method for recombinantproduction of antibodies is disclosed in U.S. Pat. No. 4,816,567.

Transformation can be by any known method for introducingpolynucleotides into a host cell. Methods for introduction ofheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,biolistic injection and direct microinjection of the DNA into nuclei. Inaddition, nucleic acid molecules may be introduced into mammalian cellsby viral vectors. Methods of transforming cells are well known in theart. See, for example, U.S. Pat. Nos. 4,399,216; 4,912,040; 4,740,461and 4,959,455.

Thus, the present invention includes recombinant methods for making ananti-TMPRSS2 antigen-binding protein, such as an antibody orantigen-binding fragment thereof of the present invention, or animmunoglobulin chain thereof, comprising (i) introducing one or morepolynucleotides (e.g., including the nucleotide sequence in any one ormore of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 or 15) encoding light and/orheavy immunoglobulin chains of the antigen-binding protein, e.g.,H1H7017N or H4H7017N, for example, wherein the polynucleotide is in avector; and/or integrated into a host cell chromosome and/or is operablylinked to a promoter; (ii) culturing the host cell (e.g., CHO or Pichiaor Pichia pastoris) under condition favorable to expression of thepolynucleotide and, (iii) optionally, isolating the antigen-bindingprotein, (e.g., antibody or fragment) or chain from the host cell and/ormedium in which the host cell is grown. When making an antigen-bindingprotein (e.g., antibody or antigen-binding fragment) comprising morethan one immunoglobulin chain, e.g., an antibody that comprises twoheavy immunoglobulin chains and two light immunoglobulin chains,co-expression of the chains in a single host cell leads to associationof the chains, e.g., in the cell or on the cell surface or outside thecell if such chains are secreted, so as to form the antigen-bindingprotein (e.g., antibody or antigen-binding fragment). The methodsinclude those wherein only a heavy immunoglobulin chain or only a lightimmunoglobulin chain (e.g., any of those discussed herein includingmature fragments and/or variable domains thereof) is expressed. Suchchains are useful, for example, as intermediates in the expression of anantibody or antigen-binding fragment that includes such a chain. Forexample, the present invention also includes anti-TMPRSS2antigen-binding proteins, such as antibodies and antigen-bindingfragments thereof, comprising a heavy chain immunoglobulin (or variabledomain thereof or comprising the CDRs thereof) encoded by apolynucleotide comprising the nucleotide sequences set forth in SEQ IDNO: 1 and a light chain immunoglobulin (or variable domain thereof orcomprising the CDRs thereof) encoded by the nucleotide sequence setforth in SEQ ID NO: 3 which are the product of such production methods,and, optionally, the purification methods set forth herein. For example,in an embodiment of the invention, the product of the method is ananti-TMPRSS2 antigen-binding protein which is an antibody or fragmentcomprising a V_(H) comprising the amino acid sequence set forth in SEQID NO: 2 and a V_(L) comprising the amino acid sequence set forth in SEQID NO: 4; or comprising a HC comprising the amino acid sequence setforth in SEQ ID NO: 17 or 19 and a LC comprising the amino acid sequenceset forth in SEQ ID NO: 18.

Eukaryotic and prokaryotic host cells, including mammalian cells, may beused as hosts for expression of an anti-TMPRSS2 antigen-binding protein.Such host cells are well known in the art and many are available fromthe American Type Culture Collection (ATCC). These host cells include,inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLacells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells,HEK-293 cells and a number of other cell lines. Mammalian host cellsinclude human, mouse, rat, dog, monkey, pig, goat, bovine, horse andhamster cells. Other cell lines that may be used are insect cell lines(e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells,bacterial cells, plant cells and fungal cells. Fungal cells includeyeast and filamentous fungus cells including, for example, Pichiapastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae,Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichialindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria,Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica,Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenulapolymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans,Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichodermareesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum,Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Thepresent invention includes an isolated host cell (e.g., a CHO cell)comprising an antigen-binding protein, such as H1H7017N; or apolynucleotide encoding such a polypeptide thereof.

The term “specifically binds” refers to those antigen-binding proteins(e.g., mAbs) having a binding affinity to an antigen, such as TMPRSS2protein (e.g., human TMPRSS2), expressed as K_(D), of at least about10⁻⁸ M (e.g., 2.81×10⁻⁹M; 9.31×10⁻⁹M; 10⁻⁹ M; 10⁻¹⁰M, 10⁻¹¹ M, or 10⁻¹²M), as measured by real-time, label free bio-layer interferometry assay,for example, at 25° C. or 37° C., e.g., an Octet® HTX biosensor, or bysurface plasmon resonance, e.g., BIACORE™, or by solution-affinityELISA. The present invention includes antigen-binding proteins thatspecifically bind to TMPRSS2 protein.

The terms “antigen-binding portion” or “antigen-binding fragment” of anantibody or antigen-binding protein, and the like, as used herein,include any naturally occurring, enzymatically obtainable, synthetic, orgenetically engineered polypeptide or glycoprotein that specificallybinds an antigen to form a complex. Non-limiting examples ofantigen-binding fragments include: (i) Fab fragments; (ii) F(ab′)2fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv(scFv) molecules; (vi) dAb fragments; and (vii) minimal recognitionunits consisting of the amino acid residues that mimic the hypervariableregion of an antibody (e.g., an isolated complementarity determiningregion (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4peptide. Other engineered molecules, such as domain-specific antibodies,single domain antibodies, domain-deleted antibodies, chimericantibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies,minibodies, nanobodies (e.g., as defined in WO08/020079 or WO09/138519)(e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein. In an embodiment of the invention, the antigen-bindingfragment comprises three or more CDRs of H1H7017N (e.g., CDR-H1, CDR-H2and CDR-H3; or CDR-L1, CDR-L2 and CDR-L3).

An antigen-binding fragment of an antibody will, in an embodiment of theinvention, comprise at least one variable domain. The variable domainmay be of any size or amino acid composition and will generally compriseat least one CDR, which is adjacent to or in frame with one or moreframework sequences. In antigen-binding fragments having a V_(H) domainassociated with a V_(L) domain, the V_(H) and V_(L) domains may besituated relative to one another in any suitable arrangement. Forexample, the variable region may be dimeric and contain V_(H)-V_(H),V_(H)-V_(L) or V_(L)-V_(L) dimers. Alternatively, the antigen-bindingfragment of an antibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

Antigen-binding proteins (e.g., antibodies and antigen-bindingfragments) may be mono-specific or multi-specific (e.g., bi-specific).Multispecific antigen-binding proteins are discussed further herein.

In specific embodiments, antibody or antibody fragments of the inventionmay be conjugated to a moiety such a ligand or a therapeutic moiety(“immunoconjugate”), such as an anti-viral drug, a second anti-influenzaantibody, or any other therapeutic moiety useful for treating a viralinfection, e.g., influenza viral infection. See below.

The present invention also provides a complex comprising an anti-TMPRSS2antigen-binding protein, e.g., antibody or antigen-binding fragment,discussed herein complexed with TMPRSS2 polypeptide or an antigenicfragment thereof and/or with a secondary antibody or antigen-bindingfragment thereof (e.g., detectably labeled secondary antibody) thatbinds specifically to the anti-TMPRSS2 antibody or fragment. In anembodiment of the invention, the antibody or fragment is in vitro (e.g.,is immobilized to a solid substrate) or is in the body of a subject. Inan embodiment of the invention, the TMPRSS2 is in vitro (e.g., isimmobilized to a solid substrate) or is on the surface of a cell or isin the body of a subject. Immobilized anti-TMRPSS2 antibodies andantigen-binding fragments thereof which are covalently linked to aninsoluble matrix material (e.g., glass or polysaccharide such as agaroseor sepharose, e.g., a bead or other particle thereof) are also part ofthe present invention; optionally, wherein the immobilized antibody iscomplexed with TMPRSS2 or antigenic fragment thereof or a secondaryantibody or fragment thereof.

“Isolated” antigen-binding proteins, antibodies or antigen-bindingfragments thereof, polypeptides, polynucleotides and vectors, are atleast partially free of other biological molecules from the cells orcell culture from which they are produced. Such biological moleculesinclude nucleic acids, proteins, other antibodies or antigen-bindingfragments, lipids, carbohydrates, or other material such as cellulardebris and growth medium. An isolated antibody or antigen-bindingfragment may further be at least partially free of expression systemcomponents such as biological molecules from a host cell or of thegrowth medium thereof. Generally, the term “isolated” is not intended torefer to a complete absence of such biological molecules or to anabsence of water, buffers, or salts or to components of a pharmaceuticalformulation that includes the antibodies or fragments.

The term “epitope” refers to an antigenic determinant (e.g., on TMPRSS2polypeptide) that interacts with a specific antigen-binding site of anantigen-binding protein, e.g., a variable region of an antibodymolecule, known as a paratope. A single antigen may have more than oneepitope. Thus, different antibodies may bind to different areas on anantigen and may have different biological effects. The term “epitope”also refers to a site on an antigen to which B and/or T cells respond.It also refers to a region of an antigen that is bound by an antibody.Epitopes may be defined as structural or functional. Functional epitopesare generally a subset of the structural epitopes and have thoseresidues that directly contribute to the affinity of the interaction.Epitopes may be linear or conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

Methods for determining the epitope of an antigen-binding protein, e.g.,antibody or fragment or polypeptide, include alanine scanning mutationalanalysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide cleavage analysis, crystallographic studies and NMRanalysis. In addition, methods such as epitope excision, epitopeextraction and chemical modification of antigens can be employed (Tomer(2000) Prot. Sci. 9: 487-496). Another method that can be used toidentify the amino acids within a polypeptide with which anantigen-binding protein (e.g., antibody or fragment or polypeptide)(e.g., coversin) interacts is hydrogen/deuterium exchange detected bymass spectrometry. In general terms, the hydrogen/deuterium exchangemethod involves deuterium-labeling the protein of interest, followed bybinding the antigen-binding protein, e.g., antibody or fragment orpolypeptide, to the deuterium-labeled protein. Next, the TMPRSS2protein/antigen-binding protein complex is transferred to water andexchangeable protons within amino acids that are protected by theantibody complex undergo deuterium-to-hydrogen back-exchange at a slowerrate than exchangeable protons within amino acids that are not part ofthe interface. As a result, amino acids that form part of theprotein/antigen-binding protein interface may retain deuterium andtherefore exhibit relatively higher mass compared to amino acids notincluded in the interface. After dissociation of the antigen-bindingprotein (e.g., antibody or fragment or polypeptide), the target proteinis subjected to protease cleavage and mass spectrometry analysis,thereby revealing the deuterium-labeled residues which correspond to thespecific amino acids with which the antigen-binding protein interacts.See, e.g., Ehring (1999) Analytical Biochemistry 267: 252-259; Engen andSmith (2001) Anal. Chem. 73: 256A-265A.

The term “competes” as used herein, refers to an antigen-binding protein(e.g., antibody or antigen-binding fragment thereof) that binds to anantigen (e.g., TMPRSS2) and inhibits or blocks the binding of anotherantigen-binding protein (e.g., antibody or antigen-binding fragmentthereof) to the antigen. The term also includes competition between twoantigen-binding proteins e.g., antibodies, in both orientations, i.e., afirst antibody that binds and blocks binding of second antibody and viceversa. In certain embodiments, the first antigen-binding protein (e.g.,antibody) and second antigen-binding protein (e.g., antibody) may bindto the same epitope. Alternatively, the first and second antigen-bindingproteins (e.g., antibodies) may bind to different, but, for example,overlapping epitopes, wherein binding of one inhibits or blocks thebinding of the second antibody, e.g., via steric hindrance. Competitionbetween antigen-binding proteins (e.g., antibodies) may be measured bymethods known in the art, for example, by a real-time, label-freebio-layer interferometry assay. In an embodiment of the invention,competition between a first and second anti-TMPRSS2 antigen-bindingprotein (e.g., antibody) is determined by measuring the ability of animmobilized first anti-TMPRSS2 antigen-binding protein (e.g., antibody)(not initially complexed with TMPRSS2 protein) to bind to solubleTMPRSS2 protein complexed with a second anti-TMPRSS2 antigen-bindingprotein (e.g., antibody). A reduction in the ability of the firstanti-TMPRSS2 antigen-binding protein (e.g., antibody) to bind to thecomplexed TMPRSS2 protein, relative to uncomplexed TMPRSS2 protein,indicates that the first and second anti-TMPRSS2 antigen-bindingproteins (e.g., antibodies) compete. The degree of competition can beexpressed as a percentage of the reduction in binding. Such competitioncan be measured using a real time, label-free bio-layer interferometryassay, e.g., on an Octet RED384 biosensor (Pall ForteBio Corp.), ELISA(enzyme-linked immunosorbent assays) or SPR (surface plasmon resonance).

Binding competition between anti-TMPRSS2 antigen-binding proteins (e.g.,monoclonal antibodies (mAbs)) can be determined using a real time,label-free bio-layer interferometry assay on an Octet RED384 biosensor(Pall ForteBio Corp.). For example, to determine competition between twoanti-human TMPRSS2 monoclonal antibodies, the anti-TMPRSS2 mAb can befirst captured onto anti-hFc antibody coated Octet biosensor tips (PallForteBio Corp., #18-5060) by submerging the tips into a solution ofanti-human TMPRSS2 mAb (subsequently referred to as “mAb1”). As apositive-control for blocking, the antibody captured biosensor tips canthen be saturated with a known blocking isotype control mAb(subsequently referred to as “blocking mAb”) by dipping into a solutionof blocking mAb. To determine if mAb2 competes with mAb1, the biosensortips can then be subsequently dipped into a co-complexed solution ofhuman TMPRSS2 polypeptide and a second anti-human TMPRSS2 mAb(subsequently referred to as “mAb2”), that had been pre-incubated for aperiod of time and binding of mAb1 to the TMPRSS2 polypeptide can bedetermined. The biosensor tips can be washed in buffer in between everystep of the experiment. The real-time binding response can be monitoredduring the course of the experiment and the binding response at the endof every step can be recorded.

For example, in an embodiment of the invention, the competition assay isconducted at 25° C. and pH about 7, e.g., 7.4, e.g., in the presence ofbuffer, salt, surfactant and a non-specific protein (e.g., bovine serumalbumin).

Typically, an antibody or antigen-binding fragment of the inventionwhich is modified in some way retains the ability to specifically bindto TMPRSS2, e.g., retains at least 10% of its TMPRSS2 binding activity(when compared to the parental antibody) when that activity is expressedon a molar basis. Preferably, an antibody or antigen-binding fragment ofthe invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% ormore of the TMPRSS2 binding affinity as the parental antibody. It isalso intended that an antibody or antigen-binding fragment of theinvention can include conservative or non-conservative amino acidsubstitutions (referred to as “conservative variants” or “functionconserved variants” of the antibody) that do not substantially alter itsbiologic activity.

A “variant” of a polypeptide, such as an immunoglobulin chain (e.g.,H1H7017N V_(H), V_(L), HC or LC), refers to a polypeptide comprising anamino acid sequence that is at least about 70-99.9% (e.g., 70, 72, 74,75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 99.5, 99.9%) identical or similar to a referencedamino acid sequence that is set forth herein (e.g., SEQ ID NO: 2, 4, 17,18 or 19); when the comparison is performed by a BLAST algorithm whereinthe parameters of the algorithm are selected to give the largest matchbetween the respective sequences over the entire length of therespective reference sequences (e.g., expect threshold: 10; word size:3; max matches in a query range: 0; BLOSUM 62 matrix; gap costs:existence 11, extension 1; conditional compositional score matrixadjustment).

A “variant” of a polynucleotide refers to a polynucleotide comprising anucleotide sequence that is at least about 70-99.9% (e.g., 70, 72, 74,75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 99.5, 99.9%) identical to a referenced nucleotidesequence that is set forth herein (e.g., SEQ ID NO: 1 or 3); when thecomparison is performed by a BLAST algorithm wherein the parameters ofthe algorithm are selected to give the largest match between therespective sequences over the entire length of the respective referencesequences (e.g., expect threshold: 10; word size: 28; max matches in aquery range: 0; match/mismatch scores: 1, −2; gap costs: linear).

Anti-TMPRSS2 antigen-binding proteins, e.g., antibodies andantigen-binding fragments thereof of the present invention, in anembodiment of the invention, include a heavy chain immunoglobulinvariable region having at least 70% (e.g., 80%, 85%, 90%, 95%, 99%)amino acid sequence identity to the amino acids set forth in SEQ ID NO:2, 17 or 19; and/or a light chain immunoglobulin variable region havingat least 70% (e.g., 80%, 85%, 90%, 95%, 99%) amino acid sequenceidentity to the amino acids set forth in SEQ ID NO: 4 or 18.

In addition, a variant anti-TMPRSS2 antigen-binding protein may includea polypeptide comprising an amino acid sequence that is set forth hereinexcept for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) mutationssuch as, for example, missense mutations (e.g., conservativesubstitutions), non-sense mutations, deletions, or insertions. Forexample, the present invention includes antigen-binding proteins whichinclude an immunoglobulin light chain variant comprising the amino acidsequence set forth in SEQ ID NO: 4 or 18 but having one or more of suchmutations and/or an immunoglobulin heavy chain variant comprising theamino acid sequence set forth in SEQ ID NO: 2, 17 or 19 but having oneor more of such mutations. In an embodiment of the invention, a variantanti-TMPRSS2 antigen-binding protein includes an immunoglobulin lightchain variant comprising CDR-L1, CDR-L2 and CDR-L3 wherein one or more(e.g., 1 or 2 or 3) of such CDRs has one or more of such mutations(e.g., conservative substitutions) and/or an immunoglobulin heavy chainvariant comprising CDR-H1, CDR-H2 and CDR-H3 wherein one or more (e.g.,1 or 2 or 3) of such CDRs has one or more of such mutations (e.g.,conservative substitutions).

The invention further provides variant anti-TMPRSS2 antigen-bindingproteins, e.g., antibodies or antigen-binding fragments thereof,comprising one or more variant CDRs (e.g., any one or more of CDR-L1,CDR-L2, CDR-L3, CDR-H1, CDR-H2 and/or CDR-H3) that are set forth hereinwith at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 99.9% sequenceidentity or similarity to, e.g., SEQ ID NO: 12, 14, 16, 6, 8 and/or 10.

Embodiments of the present invention also include variantantigen-binding proteins, e.g., anti-TMPRSS2 antibodies andantigen-binding fragments thereof, that comprise immunoglobulin V_(H)Sand V_(L)s; or HCs and LCs, which comprise an amino acid sequence having70% or more (e.g., 80%, 85%, 90%, 95%, 97% or 99%) overall amino acidsequence identity or similarity to the amino acid sequences of thecorresponding V_(H)S, V_(L)s, HCs or LCs specifically set forth herein,but wherein the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 ofsuch immunoglobulins are not variants and comprise the amino acidsequence set forth in SEQ ID NOs: 12, 14, 16, 6, 8 and 10, respectively.Thus, in such embodiments, the CDRs within variant antigen-bindingproteins are not, themselves, variants.

Conservatively modified variant anti-TMPRSS2 antibodies andantigen-binding fragments thereof are also part of the presentinvention. A “conservatively modified variant” or a “conservativesubstitution” refers to a variant wherein there is one or moresubstitutions of amino acids in a polypeptide with other amino acidshaving similar characteristics (e.g. charge, side-chain size,hydrophobicity/hydrophilicity, backbone conformation and rigidity,etc.). Such changes can frequently be made without significantlydisrupting the biological activity of the antibody or fragment. Those ofskill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity (see, e.g., Watson et al. (1987)Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224(4^(th) Ed.)). In addition, substitutions of structurally orfunctionally similar amino acids are less likely to significantlydisrupt biological activity.

Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartate and glutamate, and 7) sulfur-containingside chains: cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443 45.

Function-conservative variants of the anti-TMPRSS2 antibodies andantigen-binding fragments thereof are also part of the presentinvention. Any of the variants of the anti-TMPRSS2 antibodies andantigen-binding fragments thereof (as discussed herein) may be“function-conservative variants”. Such function-conservative variantsmay, in some cases, also be characterized as conservatively modifiedvariants. “Function-conservative variants,” as used herein, refers tovariants of the anti-TMPRSS2 antibodies or antigen-binding fragmentsthereof in which one or more amino acid residues have been changedwithout significantly altering one or more functional properties of theantibody or fragment. In an embodiment of the invention, afunction-conservative variant anti-TMPRSS2 antibody or antigen-bindingfragment thereof of the present invention comprises a variant amino acidsequence and exhibits one or more of the following functionalproperties:

-   -   Inhibits growth of influenza virus (e.g., A/Puerto Rico/08/1934        (H1N1)) in TMPRSS2-expressing cells (e.g., Calu-3 cells);    -   Binds to the surface of TMPRSS-expressing cells (e.g.,        MDCK/Tet-on), e.g., with an EC₅₀ value of 440 pM or 1.06 nM,        respectively;    -   Does not significantly bind to MDCK/Tet-on cells which do not        express TMPRSS2;    -   Binds to human TMPRSS2 with a K_(D) of about 2.81×10⁻⁹M at about        25° C.;    -   Binds to human TMPRSS2 with a K_(D) of about 9.31×10⁻⁹M at about        37° C.;    -   Binds to cynomolgous TMPRSS2 with a K_(D) of about 5.60×10⁻⁸M at        about 25° C.;    -   Binds to cynomolgous TMPRSS2 with a K_(D) of about 1.40×10⁻⁷M at        about 37° C.;    -   Limits spread of influenza virus infection (e.g., by H1_PR34;        H1_CA09; H1_Bris; H9N2 or H3N2 influenza virus) of cells, e.g.,        Calu-3, in vitro; and/or    -   Protects a mouse engineered to express the human TMPRSS2 protein        from death caused by influenza virus infection, e.g., H1N1, or        H3N2, for example, wherein the mice are infected with an        otherwise lethal dose of the virus, optionally when combined        with an anti-HA antibody.

The present invention includes a mouse engineered to express the humanTMPRSS2 protein which includes, within the mouse's body, an anti-TMPRSS2antigen-binding protein (e.g., antibody or antigen-binding fragment)such as H1H7017N and H4H7017N. See International patent applicationpublication no. WO2017/151453.

A “neutralizing” or “antagonist” anti-TMPRSS2 antigen-binding protein,e.g., antibody or antigen-binding fragment, refers to a molecule thatinhibits an activity of TMPRSS2 to any detectable degree, e.g., inhibitsprotease activity of TMPRSS2, for example, of a substrate such as HA;Cbz-Gly-Gly-Arg-AMC (Sigma), where Cbz is benzyloxycarbonyl and AMC is7-amino-4-methylcoumarin; influenza virus HA0; coronavirus S protein; orprecursor TMPRSS2 which is autocatalytically cleaved between Arg255 andIle256 and/or inhibits influenza virus entry into a cell and/or inhibitsinfluenza virus reproduction in the body of a subject.

“H1H7017N” and “H4H7017N” refer to antigen-binding proteins, such asantibodies and antigen-binding fragments thereof, that comprise theheavy chain or V_(H) (or a variant thereof) and light chain or V_(L) (ora variant thereof) as set forth below; or that comprise a V_(H) thatcomprises the CDRs thereof (CDR-H1 (or a variant thereof), CDR-H2 (or avariant thereof) and CDR-H3 (or a variant thereof)) and a V_(L) thatcomprises the CDRs thereof (CDR-L1 (or a variant thereof), CDR-L2 (or avariant thereof) and CDR-L3 (or a variant thereof)), e.g., wherein theimmunoglobulin chains, variable regions and/or CDRs comprise thespecific amino acid sequences described below.

In an embodiment of the invention, “H1H7017N” or “H4H7017N” refers to anantibody or antigen-binding fragment thereof comprising CDR-H1, CDR-H2,and CDR-H3 of an immunoglobulin heavy chain that comprises the aminoacid sequence set forth in SEQ ID NO: 2, 17 or 19 and CDR-L1, CDR-L2,and CDR-L3 of an immunoglobulin light chain that comprises the aminoacid sequence set forth in SEQ ID NO: 4 or 18.

In an embodiment of the invention, “H1H7017N” or “H4H7017N” refers to anantibody or antigen-binding fragment thereof comprising a V_(H) thatcomprises the amino acid sequence set forth in SEQ ID NO: 2; and a V_(L)that comprises the amino acid sequence set forth in SEQ ID NO: 4.

In an embodiment of the invention, “H1H7017N” refers to an antibody orantigen-binding fragment comprising a heavy chain immunoglobulin thatcomprises the amino acid sequence set forth in SEQ ID NO: 17; and alight chain immunoglobulin that comprises the amino acid sequence setforth in SEQ ID NO: 18.

In an embodiment of the invention, “H4H7017N” refers to an antibody orantigen-binding fragment comprising a heavy chain immunoglobulin thatcomprises the amino acid sequence set forth in SEQ ID NO: 19; and alight chain immunoglobulin that comprises the amino acid sequence setforth in SEQ ID NO: 18. The term “H4H7017N” also includes embodimentswherein the V_(H) is fused to a wild-type IgG4, e.g., wherein residue108 is S.

Anti-TMRPS22 Antibody or Antigen-Binding FragmentH1H7017N and H4H7017NH1H7017N and H4H7017N HeavyChain Variable Region (DNA) (SEQ ID NO: 1)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTTCCTATGGCATGCACTGGGTCCGCCAGTCTCCAGGCAAGGGGCTCGAGTGGGTGGCAGTTATATGGAATGATGGAAGTTATGTATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACATTTCCAAGAACACGCTGTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGGAGTGGGTACTTTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCAH1H7017N and H4H7017N Heavy Chain Variable Region (Polypeptide)(SEQ ID NO: 2) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQSPGKGLEWVAVIWNDGSYVYYADSVKGRFTISRDISKNTLFLQMNSLRAEDTAVYYCAREG EWVLYYFDYWGQGTLVTVSSH1H7017N and H4H7017N Light Chain Variable Region  (DNA) (SEQ ID NO: 3)GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTTGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATAAGGCGTCTACTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCGTACACTTTTGGCCAG GGGACCAAGCTGGAGATCAAAH1H7017N and H4H7017N Light Chain Variable Region  (Polypeptide)(SEQ ID NO: 4) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQ GTKLEIKH1H7017N and H4H7017N CDR-H1 (DNA) (SEQ ID NO: 5)GGA TTC ACC TTC AGT TCC TAT GGCH1H7017N and H4H7017N CDR-H1 (Polypeptide) (SEQ ID NO: 6)G F T F S S Y G (or a variant thereof having 1, 2, 3 or 4 pointmutations and/or point deletions) H1H7017N and H4H7017N CDR-H2 (DNA)(SEQ ID NO: 7) ATA TGG AAT GAT GGA AGT TAT GTAH1H7017N and H4H7017N CDR-H2 (Polypeptide) (SEQ ID NO: 8)I W N D G S Y V (or a variant thereof having 1, 2, 3 or 4 pointmutations and/or point deletions) H1H7017N and H4H7017N CDR-H3 (DNA)(SEQ ID NO: 9) GCG AGA GAG GGG GAG TGG GTA CTT TAC TAC TTT GAC TACH1H7017N and H4H7017N CDR-H3 (Polypeptide) (SEQ ID NO: 10)A R E G E W V L Y Y F D Y(or a variant thereof having 1, 2, 3 or 4 pointmutations and/or point deletions) H1H7017N and H4H7017N CDR-L1 (DNA)(SEQ ID NO: 11) CAG AGT ATT AGT AGC TGGH1H7017N and H4H7017N CDR-L1 (Polypeptide) (SEQ ID NO: 12) Q S I S S W(or a variant thereof having 1, 2, 3 or 4 pointmutations and/or point deletions) H1H7017N and H4H7017N CDR-L2 (DNA)(SEQ ID NO: 13) AAG GCG TCT H1H7017N and H4H7017N CDR-L2 (Polypeptide)(SEQ ID NO: 14) K A S (or a variant thereof having a point mutation and/or point deletion) H1H7017N and H4H7017N CDR-L3 (DNA) (SEQ ID NO: 15)CAA CAG TAT AAT AGT TAT TCG TAC ACTH1H7017N and H4H7017N CDR-L3 (Polypeptide) (SEQ ID NO: 16)Q Q Y N S Y S Y T (or a variant thereof having 1, 2, 3 or 4 pointmutations and/or point deletions) H1H7017NFull length heavy chain-human IgG1 (SEQ ID NO: 17)QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQSPGKGLEWVAVIWNDGSYVYYADSVKGRFTISRDISKNTLFLQMNSLRAEDTAVYYCAREGEWVLYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFull length light chain-human Kappa (SEQ ID NO: 18)DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECH4H7017N Full length heavy chain-human IgG4 (S108P) (SEQ ID NO: 19)QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQSPGKGLEWVAVIWNDGSYVYYADSVKGRFTISRDISKNTLFLQMNSLRAEDTAVYYCAREGEWVLYYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKFull length light chain-human Kappa (SEQ ID NO: 18)DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

Antibodies and antigen-binding fragments of the present inventioncomprise immunoglobulin chains including the amino acid sequences setforth herein as well as cellular and in vitro post-translationalmodifications to the antibody. For example, the present inventionincludes antibodies and antigen-binding fragments thereof thatspecifically bind to TMPRSS2 comprising heavy and/or light chain aminoacid sequences set forth herein (e.g., CDR-H1, CDR-H2, CDR-H3, CDR-L1,CDR-L2 and/or CDR-L3) as well as antibodies and fragments wherein one ormore amino acid residues is glycosylated, one or more Asn residues isdeamidated, one or more residues (e.g., Met, Trp and/or His) isoxidized, the N-terminal Gln is pyroglutamate (pyroE) and/or theC-terminal Lysine is missing.

The present invention provides a vessel (e.g., a plastic or glass vial,e.g., with a cap or a chromatography column, hollow bore needle or asyringe cylinder) comprising an anti-TMPRSS2 antigen-binding protein ofthe present invention, e.g., H1H7017N or H4H7017N.

The present invention also provides an injection device comprising oneor more antigen-binding proteins (e.g., antibody or antigen-bindingfragment) that bind specifically to TMPRSS2, e.g., H4H7017N or H1H7017N,or a pharmaceutical composition thereof. The injection device may bepackaged into a kit. An injection device is a device that introduces asubstance into the body of a subject via a parenteral route, e.g.,intramuscular, subcutaneous or intravenous. For example, an injectiondevice may be a syringe (e.g., pre-filled with the pharmaceuticalcomposition, such as an auto-injector) which, for example, includes acylinder or barrel for holding fluid to be injected (e.g., comprisingthe antibody or fragment or a pharmaceutical composition thereof), aneedle for piecing skin and/or blood vessels for injection of the fluid;and a plunger for pushing the fluid out of the cylinder and through theneedle bore. In an embodiment of the invention, an injection device thatcomprises an antigen-binding protein, e.g., an antibody orantigen-binding fragment thereof, from a combination of the presentinvention, or a pharmaceutical composition thereof is an intravenous(IV) injection device. Such a device can include the antigen-bindingprotein or a pharmaceutical composition thereof in a cannula ortrocar/needle which may be attached to a tube which may be attached to abag or reservoir for holding fluid (e.g., saline) introduced into thebody of the subject through the cannula or trocar/needle. The antibodyor fragment or a pharmaceutical composition thereof may, in anembodiment of the invention, be introduced into the device once thetrocar and cannula are inserted into the vein of a subject and thetrocar is removed from the inserted cannula. The IV device may, forexample, be inserted into a peripheral vein (e.g., in the hand or arm);the superior vena cava or inferior vena cava, or within the right atriumof the heart (e.g., a central IV); or into a subclavian, internaljugular, or a femoral vein and, for example, advanced toward the heartuntil it reaches the superior vena cava or right atrium (e.g., a centralvenous line). In an embodiment of the invention, an injection device isan autoinjector; a jet injector or an external infusion pump. A jetinjector uses a high-pressure narrow jet of liquid which penetrate theepidermis to introduce the antibody or fragment or a pharmaceuticalcomposition thereof to a subject's body. External infusion pumps aremedical devices that deliver the antibody or fragment or apharmaceutical composition thereof into a subject's body in controlledamounts. External infusion pumps may be powered electrically ormechanically. Different pumps operate in different ways, for example, asyringe pump holds fluid in the reservoir of a syringe, and a moveablepiston controls fluid delivery, an elastomeric pump holds fluid in astretchable balloon reservoir, and pressure from the elastic walls ofthe balloon drives fluid delivery. In a peristaltic pump, a set ofrollers pinches down on a length of flexible tubing, pushing fluidforward. In a multi-channel pump, fluids can be delivered from multiplereservoirs at multiple rates.

The present invention further provides methods for administering ananti-TMPRSS2 antigen-binding protein of the present invention, e.g.,H4H7017N or H1H7017N, comprising introducing the antigen-binding proteininto the body of a subject (e.g., a human). For example, the methodcomprises piercing the body of the subject with a needle of a syringeand injecting the antigen-binding protein into the body of the subject,e.g., into the vein, artery, tumor, muscular tissue or subcutis of thesubject.

Preparation of Human Antibodies

Methods for generating human antibodies in transgenic mice are known inthe art. Any such known methods can be used in the context of thepresent invention to make human antibodies that specifically bind toTMPRSS2. An immunogen comprising any one of the following can be used togenerate antibodies to TMPRSS2. In certain embodiments of the invention,the antibodies of the invention are obtained from mice immunized with afull length, native TMPRSS2, or with a live attenuated or inactivatedvirus, or with DNA encoding the protein or fragment thereof.Alternatively, the TMPRSS2 protein or a fragment thereof may be producedusing standard biochemical techniques and modified and used asimmunogen. In one embodiment of the invention, the immunogen is arecombinantly produced TMPRSS2 protein or fragment thereof. In certainembodiments of the invention, the immunogen may be a TMPRSS2 polypeptidevaccine. In certain embodiments, one or more booster injections may beadministered. In certain embodiments, the immunogen may be a recombinantTMPRSS2 polypeptide expressed in E. coli or in any other eukaryotic ormammalian cells such as Chinese hamster ovary (CHO) cells.

Using VELOCIMMUNE® technology (see, for example, U.S. Pat. No.6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other knownmethod for generating monoclonal antibodies, high affinity chimericantibodies to TMPRSS2 can be initially isolated having a human variableregion and a mouse constant region. The VELOCIMMUNE® technology involvesgeneration of a transgenic mouse having a genome comprising human heavyand light chain variable regions operably linked to endogenous mouseconstant region loci such that the mouse produces an antibody comprisinga human variable region and a mouse constant region in response toantigenic stimulation. The DNA encoding the variable regions of theheavy and light chains of the antibody are isolated and operably linkedto DNA encoding the human heavy and light chain constant regions. TheDNA is then expressed in a cell capable of expressing the fully humanantibody.

Generally, a VELOCIMMUNE® mouse is challenged with the antigen ofinterest, and lymphatic cells (such as B-cells) are recovered from themice that express antibodies. The lymphatic cells may be fused with amyeloma cell line to prepare immortal hybridoma cell lines, and suchhybridoma cell lines are screened and selected to identify hybridomacell lines that produce antibodies specific to the antigen of interest.DNA encoding the variable regions of the heavy chain and light chain maybe isolated and linked to desirable isotypic constant regions of theheavy chain and light chain. Such an antibody protein may be produced ina cell, such as a CHO cell. Alternatively, DNA encoding theantigen-specific chimeric antibodies or the variable domains of thelight and heavy chains may be isolated directly from antigen-specificlymphocytes.

Initially, high affinity chimeric antibodies are isolated having a humanvariable region and a mouse constant region. As in the experimentalsection below, the antibodies are characterized and selected fordesirable characteristics, including affinity, selectivity, epitope,etc. The mouse constant regions are replaced with a desired humanconstant region to generate the fully human antibody of the invention,for example wild-type or modified IgG1 or IgG4. While the constantregion selected may vary according to specific use, high affinityantigen-binding and target specificity characteristics reside in thevariable region.

Anti-TMPRSS2 Antibodies Comprising Fc Variants

According to certain embodiments of the present invention, anti-TMPRSS2antigen-binding proteins, e.g., antibodies or antigen-binding fragments,are provided comprising an Fc domain comprising one or more mutations,which, for example, enhance or diminish antibody binding to the FcRnreceptor, e.g., at acidic pH as compared to neutral pH. For example, thepresent invention includes anti-TMPRSS2 antibodies comprising a mutationin the C_(H)2 or a C_(H)3 region of the Fc domain, wherein themutation(s) increases the affinity of the Fc domain to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0). Such mutations may result in an increase in serum half-lifeof the antibody when administered to an animal. Non-limiting examples ofsuch Fc modifications include, e.g., a modification at position 250(e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T),254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification atposition 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., A, W,H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I(e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K)and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y,254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Qand M428L); and a 307 and/or 308 modification (e.g., 308F or 308P). Inyet another embodiment, the modification comprises a 265A (e.g., D265A)and/or a 297A (e.g., N297A) modification.

For example, the present invention includes anti-TMPRSS2 antigen-bindingproteins, e.g., antibodies or antigen-binding fragments, comprising anFc domain comprising one or more pairs or groups of mutations selectedfrom the group consisting of: 250Q and 248L (e.g., T250Q and M248L);252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g.,M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H(e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A,380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g.,H433K and N434F).

Anti-TMPRSS antigen-binding proteins, e.g., antibodies andantigen-binding fragments thereof, that comprise a V_(H) and/or V_(L) asset forth herein comprising any possible combinations of the foregoingFc domain mutations, are contemplated within the scope of the presentinvention.

The present invention also includes anti-TMPRSS2 antigen-bindingproteins, antibodies or antigen-binding fragments, comprising a V_(H)set forth herein and a chimeric heavy chain constant (C_(H)) region,wherein the chimeric C_(H) region comprises segments derived from theC_(H) regions of more than one immunoglobulin isotype. For example, theantibodies of the invention may comprise a chimeric C_(H) regioncomprising part or all of a C_(H)2 domain derived from a human IgG1,human IgG2 or human IgG4 molecule, combined with part or all of a C_(H)3domain derived from a human IgG1, human IgG2 or human IgG4 molecule.According to certain embodiments, the antibodies of the inventioncomprise a chimeric C_(H) region having a chimeric hinge region. Forexample, a chimeric hinge may comprise an “upper hinge” amino acidsequence (amino acid residues from positions 216 to 227 according to EUnumbering) derived from a human IgG1, a human IgG2 or a human IgG4 hingeregion, combined with a “lower hinge” sequence (amino acid residues frompositions 228 to 236 according to EU numbering) derived from a humanIgG1, a human IgG2 or a human IgG4 hinge region. According to certainembodiments, the chimeric hinge region comprises amino acid residuesderived from a human IgG1 or a human IgG4 upper hinge and amino acidresidues derived from a human IgG2 lower hinge. An antibody comprising achimeric C_(H) region as described herein may, in certain embodiments,exhibit modified Fc effector functions without adversely affecting thetherapeutic or pharmacokinetic properties of the antibody. (See, e.g.,WO2014/022540).

Immunoconjugates

The invention encompasses an anti-TMPRSS2 antigen-binding proteins,e.g., antibodies or antigen-binding fragments, conjugated to anothermoiety, e.g., a therapeutic moiety (an “immunoconjugate”), such as atoxoid or an anti-viral drug to treat influenza virus infection. In anembodiment of the invention, an anti-TMPRSS2 antibody or fragment isconjugated to any of the further therapeutic agents set forth herein. Asused herein, the term “immunoconjugate” refers to an antigen-bindingprotein, e.g., an antibody or antigen-binding fragment, which ischemically or biologically linked to a radioactive agent, a cytokine, aninterferon, a target or reporter moiety, an enzyme, a peptide or proteinor a therapeutic agent. The antigen-binding protein may be linked to theradioactive agent, cytokine, interferon, target or reporter moiety,enzyme, peptide or therapeutic agent at any location along the moleculeso long as it is able to bind its target (TMPRSS2). Examples ofimmunoconjugates include antibody-drug conjugates and antibody-toxinfusion proteins. In one embodiment of the invention, the agent may be asecond, different antibody that binds specifically to TMPRSS2. The typeof therapeutic moiety that may be conjugated to the anti-TMPRSS2antigen-binding protein (e.g., antibody or fragment) will take intoaccount the condition to be treated and the desired therapeutic effectto be achieved. See, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, Monoclonal Antibodies AndCancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc.1985); Hellstrom et al., “Antibodies For Drug Delivery”, Controlled DrugDelivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker,Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In CancerTherapy: A Review”, Monoclonal Antibodies 1984: Biological And ClinicalApplications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis,Results, And Future Prospective Of The Therapeutic Use Of RadiolabeledAntibody In Cancer Therapy”, Monoclonal Antibodies For Cancer DetectionAnd Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985),and Thorpe et al., “The Preparation And Cytotoxic Properties OfAntibody-Toxin Conjugates”, Immunol. Rev., 62: 119-58 (1982).

Multi-Specific Antibodies

The present invention includes anti-TMPRSS2 antigen-binding proteins,e.g., antibodies and antigen-binding fragments thereof, as well asmethods of use thereof and methods of making such antigen-bindingproteins. The term “anti-TMPRSS2” antigen-binding proteins, e.g.,antibodies or antigen-binding fragments, includes multispecific (e.g.,bispecific or biparatopic) molecules that include at least one firstantigen-binding domain that specifically binds to TMPRSS2 (e.g., anantigen-binding domain from H1H7017N or H4H7017N) and at least onesecond antigen-binding domain that binds to a different antigen or to anepitope in TMPRSS2 which is different from that of the firstantigen-binding domain (e.g., influenza HA such as an antigen-bindingdomain from H1H14611N2, H1H14612N2 or H1H11729P). In an embodiment ofthe invention, the first and second epitopes overlap. In anotherembodiment of the invention, the first and second epitopes do notoverlap. For example, in an embodiment of the invention, a multispecificantibody is a bispecific IgG antibody (e.g., IgG1 or IgG4) that includesa first antigen-binding domain that binds specifically to TMPRSS2including the heavy and light immunoglobulin chain of H1H7017N orH4H7017N, and a second antigen-binding domain that binds specifically toinfluenza HA (comprising a different light and heavy immunoglobulinchain such as from H1H14611N2, H1H14612N2 or H1H11729P).

“H1H7017N” includes a multispecific molecules, e.g., antibodies orantigen-binding fragments, that include the HCDRs and LCDRs, V_(H) andV_(L), or HC and LC of H1H7017N (including variants thereof as set forthherein).

“H4H7017N” includes a multispecific molecules, e.g., antibodies orantigen-binding fragments, that include the HCDRs and LCDRs, V_(H) andV_(L), or HC and LC of H4H7017N (including variants thereof as set forthherein).

In an embodiment of the invention, an antigen-binding domain that bindsspecifically to TMPRSS, which may be included in a multispecificmolecule, comprises:

(1)

(i) a heavy chain variable domain sequence that comprises CDR-H1comprising the amino acid sequence set forth in SEQ ID NO: 6, CDR-H2comprising the amino acid sequence set forth in SEQ ID NO: 8, and CDR-H3comprising the amino acid sequence set forth in SEQ ID NO: 10, and

(ii) a light chain variable domain sequence that comprises CDR-L1comprising the amino acid sequence set forth in SEQ ID NO: 12, CDR-L2comprising the amino acid sequence set forth in SEQ ID NO: 14, andCDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 16;

or,

(2)

(i) a heavy chain variable domain sequence comprising the amino acidsequence set forth in SEQ ID NO: 2, and

(ii) a light chain variable domain sequence comprising the amino acidsequence set forth in SEQ ID NO: 4;

or,

(3)

(i) a heavy chain immunoglobulin sequence comprising the amino acidsequence set forth in SEQ ID NO: 17 or 19, and

(ii) a light chain immunoglobulin sequence comprising the amino acidsequence set forth in SEQ ID NO: 18.

In an embodiment of the invention, the multispecific antibody orfragment includes more than two different binding specificities (e.g., atrispecific molecule), for example, one or more additionalantigen-binding domains which are the same or different from the firstand/or second antigen-binding domain.

In an embodiment of the invention, a multispecific molecule comprises,in addition to an antigen-binding site that bind specifically toTMPRSS2, an antigen-binding site that binds specifically to influenza HAtaken from an antibody selected from the group consisting of:

H1H14611N2; H1H14612N2; H1H11723P; H1H11729P; H1H11820N; H1H11829N;H1H11829N2; H2aM11829N; H2M11830N; H1H11830N2; H1H11903N; H1H14571N;H2a14571N; H1H11704P; H1H11711P; H1H11714P; H1H11717P; H1H11724P;H1H11727P; H1H11730P2; H1H11731P2; H1H11734P2; H1H11736P2; H1H11742P2;H1H11744P2; H1H11745P2; H1H11747P2; H1H11748P2; H1H17952B; H1H17953B;H1H17954B; H1H17955B; H1H17956B; H1H17957B; H1H17958B; H1H17959B;H1H17960B; H1H17961B; H1H17962B; H1H17963B; H1H17964B; H1H17965B;H1H17966B; H1H17967B; H1H17968B; H1H17969B; H1H17970B; H1H17971B;H1H17972B; H1H17973B; H1H17974B; H1H17975B; H1H17976B; H1H17977B;H1H17978B; H1H17979B; H1H17980B; H1H17981B; H1H17982B; H1H17983B;H1H17984B; H1H17985B; H1H17986B; H1H17987B; H1H17988B; H1H17989B;H1H17990B; H1H17991B; H1H17992B; H1H17993B; H1H17994B; H1H17995B;H1H17996B; H1H17997B; H1H17998B; H1H17999B; H1H18000B; H1H18001B;H1H18002B; H1H18003B; H1H18004B; H1H18005B; H1H18006B; H1H18007B;H1H18008B; H1H18009B; H1H18010B; H1H18011B; H1H18012B; H1H18013B;H1H18014B; H1H18015B; H1H18016B; H1H18017B; H1H18018B; H1H18019B;H1H18020B; H1H18021B; H1H18022B; H1H18023B; H1H18024B; H1H18025B;H1H18026B; H1H18027B; H1H18028B; H1H18029B; H1H18030B; H1H18031B;H1H18032B; H1H18033B; H1H18034B; H1H18035B; H1H18037B; H1H18038B;H1H18039B; H1H18040B; H1H18041B; H1H18042B; H1H18043B; H1H18044B;H1H18045B; H1H18046B; H1H18047B; H1H18048B; H1H18049B; H1H18051B;H1H18052B; H1H18053B; H1H18054B; H1H18055B; H1H18056B; H1H18057B;H1H18058B; H1H18059B; H1H18060B; H1H18061B; H1H18062B; H1H18063B;H1H18064B; H1H18065B; H1H18066B; H1H18067B; H1H18068B; H1H18069B;H1H18070B; H1H18071B; H1H18072B; H1H18073B; H1H18074B; H1H18075B;H1H18076B; H1H18077B; H1H18078B; H1H18079B; H1H18080B; H1H18081B;H1H18082B; H1H18083B; H1H18084B; H1H18085B; H1H18086B; H1H18087B;H1H18088B; H1H18089B; H1H18090B; H1H18091B; H1H18092B; H1H18093B;H1H18094B; H1H18095B; H1H18096B; H1H18097B; H1H18098B; H1H18099B;H1H18100B; H1H18101B; H1H18102B; H1H18103B; H1H18104B; H1H18105B;H1H18107B; H1H18108B; H1H18109B; H1H18110B; H1H18111B; H1H18112B;H1H18113B; H1H18114B; H1H18115B; H1H18116B; H1H18117B; H1H18118B;H1H18119B; H1H18120B; H1H18121B; H1H18122B; H1H18123B; H1H18124B;H1H18125B; H1H18126B; H1H18127B; H1H18128B; H1H18129B; H1H18130B;H1H18131B; H1H18132B; H1H18133B; H1H18134B; H1H18135B; H1H18136B;H1H18137B; H1H18138B; H1H18139B; H1H18140B; H1H18141B; H1H18142B;H1H18143B; H1H18144B; H1H18145B; H1H18146B; H1H18147B; H1H18148B;H1H18149B; H1H18150B; H1H18151B; H1H18152B; H1H18153B; H1H18154B;H1H18155B; H1H18156B; H1H18157B; H1H18158B; H1H18159B; H1H18160B;H1H18161B; H1H18162B; H1H18163B; H1H18164B; H1H18165B; H1H18166B;H1H18167B; H1H18168B; H1H18169B; H1H18170B; H1H18171B; H1H18172B;H1H18173B; H1H18174B; H1H18175B; H1H18176B; H1H18177B; H1H18178B;H1H18179B; H1H18180B; H1H18181B; H1H18182B; H1H18183B; H1H18184B;H1H18185B; H1H18186B; H1H18187B; H1H18188B; H1H18189B; H1H18190B;H1H18191B; H1H18192B; H1H18193B; H1H18194B; H1H18195B; H1H18196B;H1H18197B; H1H18198B; H1H18199B; H1H18200B; H1H18201B; H1H18202B;H1H18203B; H1H18204B; H1H18205B; H1H18206B; H1H18207B; H1H18208B;H1H18209B; H1H18210B; H1H18211B; H1H18212B; H1H18213B; H1H18214B;H1H18216B; H1H18217B; H1H18218B; H1H18219B; H1H18220B; H1H18221B;H1H18222B; H1H18223B; H1H18224B; H1H18225B; H1H18226B; H1H18227B;H1H18228B; H1H18229B; H1H18230B; H1H18231B; H1H18232B; H1H18233B;H1H18234B; H1H18235B; H1H18236B; H1H18237B; H1H18238B; H1H18239B;H1H18240B; H1H18241B; H1H18242B; H1H18243B; H1H18244B; H1H18245B;H1H18246B; H1H18247B; H1H18248B; H1H18249B; H1H18250B; H1H18251B;H1H18252B; H1H18253B; H1H18254B; H1H18255B; H1H18256B; H1H18257B;H1H18258B; H1H18259B; H1H18261B; H1H18262B; H1H18263B; H1H18264B;H1H18265B; H1H18266B; H1H18267B; H1H18268B; H1H18269B; H1H18270B;H1H18271B; H1H18272B; H1H18274B; H1H18275B; H1H18276B; H1H18277B;H1H18278B; H1H18279B; H1H18280B; H1H18281B; H1H18282B; H1H18283B;H1H18284B; H1H18285B; H1H18286B; H1H18287B; H1H18288B; H1H18289B;H1H18290B; H1H18291B; H1H18292B; H1H18293B; H1H18294B; H1H18295B;H1H18297B; H1H18298B; H1H18299B; H1H18300B; H1H18301B; H1H18302B;H1H18303B; H1H18304B; H1H18305B; H1H18306B; H1H18307B; H1H18308B;H1H18309B; H1H18310B; H1H18311B; H1H18312B; H1H18313B; H1H18314B;H1H18315B; H1H18316B; H1H18317B; H1H18318B; H1H18319B; H1H18320B;H1H18321B; H1H18322B; H1H18323B; H1H18324B; H1H18325B; H1H18326B;H1H18327B; H1H18328B; H1H18329B; H1H18330B; H1H18331B; H1H18332B;H1H18333B; H1H18334B; and H1H18335B; as set forth in Internationalpatent application publication no. WO2016/100807 (e.g., the CDR-Hs,V_(H) or heavy chain thereof; and the CDR-Ls, V_(L) or light chainthereof).

In an embodiment of the invention, a multispecific molecule comprises,in addition to an antigen-binding site that binds specifically toTMPRSS2, an antigen-binding site that binds specifically to influenzaGroup II HA protein, e.g., which comprises V_(H) and V_(L) of H1H14611N2(e.g., SEQ ID Nos: 24 and 28); or a heavy chain immunoglobulincomprising CDR-H1, CDR-H2 and CDR-H3 of H1H14611N2 (e.g., SEQ ID NOs:25-27) and a light chain immunoglobulin comprising CDR-L1, CDR-L2 andCDR-L3 of H1H14611N2 (e.g., SEQ ID NOs: 29-31).

In an embodiment of the invention, a multispecific molecule comprises,in addition to an antigen-binding site that bind specifically toTMPRSS2, an antigen-binding site that binds specifically to influenzaGroup II HA protein, e.g., which comprises V_(H) and V_(L) of H1H14612N2(e.g., SEQ ID Nos: 40 and 44); or a heavy chain immunoglobulincomprising CDR-H1, CDR-H2 and CDR-H3 of H1H14612N2 (e.g., SEQ ID NOs:41-43) and a light chain immunoglobulin comprising CDR-L1, CDR-L2 andCDR-L3 of H1H14612N2 (e.g., SEQ ID NOs: 45-47).

In an embodiment of the invention, a multispecific molecule comprises,in addition to an antigen-binding site that bind specifically toTMPRSS2, an antigen-binding site that binds specifically to influenzaGroup I HA protein, e.g., which comprises V_(H) and V_(L) of H1H11729P(e.g., SEQ ID Nos: 32 and 36); or a heavy chain immunoglobulincomprising CDR-H1, CDR-H2 and CDR-H3 of H1H11729P (e.g., SEQ ID NOs:33-35) and a light chain immunoglobulin comprising CDR-L1, CDR-L2 andCDR-L3 of H1H11729P (e.g., SEQ ID NOs: 37-39).

In one embodiment of the invention, a bispecific antigen-bindingfragment comprises a first scFv (e.g., comprising V_(H) and V_(L) ofH1H7017N or H4H7017N) having binding specificity for a first epitope(e.g., TMPRSS2) and a second scFv (e.g., comprising V_(H) and V_(L) ofan anti-influenza HA antibody) having binding specificity for a second,different epitope. For example, in an embodiment of the invention, thefirst and second scFv are tethered with a linker, e.g., a peptide linker(e.g., a GS linker such as (GGGGS)_(n) (SEQ ID NO: 48) wherein n is, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). Other bispecificantigen-binding fragments include an F(ab)₂ of a bispecific IgG antibodywhich comprises the heavy and light chain CDRs of H1H7017N or H4H7017Nand of another antibody that binds to a different epitope.

Therapeutic Methods

The present invention provides methods for treating or preventing viralinfection or cancer (e.g., prostate cancer) by administering atherapeutically effective amount of anti-TMPRSS2 antigen-bindingprotein, e.g., antibody or antigen-binding fragment, (e.g., H1H7017N orH4H7017N) to a subject (e.g., a human) in need of such treatment orprevention.

Influenza virus infection may be treated or prevented, in a subject, byadministering an anti-TMPRSS2 antigen-binding protein of the presentinvention to a subject. The influenza viruses are classified into typesA, B and C on the basis of their core proteins. The subtypes ofinfluenza A viruses are determined by envelope glycoproteins possessingeither hemagglutinin (HA) or neuraminidase (NA) activity. There areseveral HA subtypes (e.g., HAL HA2, HA3, HA4, HA5, HA6, HA7, HA8, HA9,HA10, HA11, HA12, HA13, HA14, HA15, HA16, HA17 or HA18—these subtypesmay be designated as H1, H2, H3, etc.) and NA subtypes (e.g., NA1, NA2,NA3, NA4, NA5, NA6, NA7, NA8, NA9, NA10 or NA11—these subtypes may bedesignated as N1, N2, N3, etc.) of influenza A viruses which are used todesignate influenza A subtype. For example, Influenza A virus H1N1 andH3N2 are commonly known human pathogens. Humans are commonly infected byviruses of the subtypes H1, H2 or H3, and N1 or N2. The presentinvention includes methods for treating or preventing infection with aninfluenza virus subtype discussed herein. Multispecific antibodies andantigen-binding fragments thereof that bind to TMPRSS2, in an embodimentof the invention, also bind to HA/and/or to NA, e.g., of a subtype setforth herein.

-   -   An effective or therapeutically effective dose of anti-TMPRSS2        antigen-binding protein, e.g., antibody or antigen-binding        fragment (e.g., H1H7017N or H4H7017N), for treating or        preventing a viral infection refers to the amount of the        antibody or fragment sufficient to alleviate one or more signs        and/or symptoms of the infection in the treated subject, whether        by inducing the regression or elimination of such signs and/or        symptoms or by inhibiting the progression of such signs and/or        symptoms. The dose amount may vary depending upon the age and        the size of a subject to be administered, target disease,        conditions, route of administration, and the like. In an        embodiment of the invention, an effective or therapeutically        effective dose of antibody or antigen-binding fragment thereof        of the present invention, for treating or preventing viral        infection, e.g., in an adult human subject, is about 0.01 to        about 200 mg/kg, e.g., up to about 150 mg/kg. In an embodiment        of the invention, the dosage is up to about 10.8 or 11 grams        (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 grams).        Depending on the severity of the infection, the frequency and        the duration of the treatment can be adjusted. In certain        embodiments, the antigen-binding protein of the present        invention can be administered at an initial dose, followed by        one or more secondary doses. In certain embodiments, the initial        dose may be followed by administration of a second or a        plurality of subsequent doses of antibody or antigen-binding        fragment thereof in an amount that can be approximately the same        or less than that of the initial dose, wherein the subsequent        doses are separated by at least 1 day to 3 days; at least one        week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at        least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8        weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks;        or at least 14 weeks.    -   As used herein, the term “subject” refers to a mammal (e.g.,        rat, mouse, cat, dog, cow, sheep, horse, goat, rabbit),        preferably a human, for example, in need of prevention and/or        treatment of a disease or disorder such as viral infection or        cancer. The subject may have a viral infection, e.g., an        influenza infection, or be predisposed to developing an        infection. Subjects predisposed to developing an infection, or        subjects who may be at elevated risk for contracting an        infection (e.g., of influenza virus), include subjects with        compromised immune systems because of autoimmune disease,        subjects receiving immunosuppressive therapy (for example,        following organ transplant), subjects afflicted with human        immunodeficiency syndrome (HIV) or acquired immune deficiency        syndrome (AIDS), subjects with forms of anemia that deplete or        destroy white blood cells, subjects receiving radiation or        chemotherapy, or subjects afflicted with an inflammatory        disorder. Additionally, subjects of very young (e.g., 5 years of        age or younger) or old age (e.g., 65 years of age or older) are        at increased risk. Moreover, a subject may be at risk of        contracting a viral infection due to proximity to an outbreak of        the disease, e.g. subject resides in a densely-populated city or        in close proximity to subjects having confirmed or suspected        infections of a virus, or choice of employment, e.g. hospital        worker, pharmaceutical researcher, traveler to infected area, or        frequent flier.    -   “Treat” or “treating” means to administer an anti-TMPRSS2        antigen-binding protein, e.g., antibody or antigen-binding        fragment of the present invention (e.g., H1H7017N or H4H7017N),        to a subject having one or more signs or symptoms of a disease        or infection, e.g., viral infection, for which the        antigen-binding protein is effective when administered to the        subject at an effective or therapeutically effective amount or        dose (as discussed herein).    -   The present invention also encompasses prophylactically        administering an anti-TMPRSS2 antigen-binding protein, e.g.,        antibody or antigen-binding fragment thereof of the present        invention (e.g., H1H7017N or H4H7017N), to a subject who is at        risk of viral infection so as to prevent such infection. Passive        antibody-based immunoprophylaxis has proven an effective        strategy for preventing subject from viral infection. See e.g.,        Berry et al., Passive broad-spectrum influenza        immunoprophylaxis. Influenza Res Treat. 2014; 2014: 267594. Epub        2014 Sep. 22; and Jianqiang et al., Passive immune        neutralization strategies for prevention and control of        influenza A infections, Immunotherapy. 2012 February; 4(2):        175-186; Prabhu et al., Antivir Ther. 2009; 14(7):911-21,        Prophylactic and therapeutic efficacy of a chimeric monoclonal        antibody specific for H5 hemagglutinin against lethal H5N1        influenza. “Prevent” or “preventing” means to administer an        anti-TMPRSS2 antigen-binding protein, e.g., antibody or        antigen-binding fragment of the present invention (e.g.,        H1H7017N or H4H7017N), to a subject to inhibit the manifestation        of a disease or infection (e.g., viral infection) in the body of        a subject, for which the antigen-binding protein is effective        when administered to the subject at an effective or        therapeutically effective amount or dose (as discussed herein).

In an embodiment of the invention, a sign or symptom of a viralinfection in a subject is survival or proliferation of virus in the bodyof the subject, e.g., as determined by viral titer assay (e.g.,influenza virus propagation in embryonated chicken eggs or influenzavirus hemagglutination assay). Other signs and symptoms of viralinfection are discussed herein.

The present invention provides a method for treating or preventing viralinfection (e.g., influenza virus or corona virus infection) or forinducing the regression or elimination or inhibiting the progression ofat least one sign or symptom of viral infection such as:

-   -   Fever or feeling feverish/chills;    -   Cough;    -   Sore throat;    -   Runny or stuffy nose;    -   Sneezing;    -   Muscle or body aches;    -   Headaches;    -   Fatigue (tiredness);    -   vomiting;    -   diarrhea;    -   respiratory tract infection;    -   chest discomfort;    -   shortness of breath;    -   bronchitis; and/or    -   pneumonia,        which sign or symptom is secondary to viral infection, in a        subject in need thereof (e.g., a human), by administering a        therapeutically effective amount of anti-TMPRSS2 antigen-binding        protein (e.g., H1H7017N or H4H7017N) to the subject, for        example, by injection of the protein into the body of the        subject.

The present invention also includes methods for treating or preventingcancer, e.g., metastatic cancer, e.g., prostate cancer (e.g., which ischaracterized by expression of a TMPRSS2:ERG fusion), colon cancer, lungcancer, pancreas cancer, urinary tract cancer, breast cancer, ovariancancer, prostate adenocarcinoma, renal cell carcinoma, colorectaladenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma and/orpleural mesothelioma, in a subject, by administering a therapeuticallyeffective amount of TMPRSS2 antigen-binding protein (e.g., H1H7017N orH4H7017N) to the subject, for example, by injection of the protein intothe body of the subject. In an embodiment of the invention, the subjectis also administered the TMPRSS2 antigen-binding protein in associationwith a further therapeutic agent, for example, an anti-cancertherapeutic agent. In an embodiment of the invention, the cancer is atumor whose cells express TMPRSS2 or a variant thereof.

Combinations and Pharmaceutical Compositions

To prepare pharmaceutical compositions of the anti-TMPRSS2antigen-binding proteins, e.g., antibodies and antigen-binding fragmentsthereof (e.g., H1H7017N or H4H7017N), antigen-binding protein is admixedwith a pharmaceutically acceptable carrier or excipient. See, e.g.,Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: NationalFormulary, Mack Publishing Company, Easton, Pa. (1984); Hardman, et al.(2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science andPractice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.;Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: ParenteralMedications, Marcel Dekker, N.Y.; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, N.Y.; Lieberman, etal. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, MarcelDekker, N.Y.; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety,Marcel Dekker, Inc., New York, N.Y. In an embodiment of the invention,the pharmaceutical composition is sterile. Such compositions are part ofthe present invention.

The scope of the present invention includes desiccated, e.g.,freeze-dried, compositions comprising an anti-TMPRSS2 antigen-bindingproteins, e.g., antibody or antigen-binding fragment thereof (e.g.,H1H7017N or H4H7017N), or a pharmaceutical composition thereof thatincludes a pharmaceutically acceptable carrier but substantially lackswater.

In a further embodiment of the invention, a further therapeutic agentthat is administered to a subject in association with an anti-TMPRSS2antigen-binding protein, e.g., antibody or antigen-binding fragmentthereof (e.g., H1H7017N or H4H7017N), disclosed herein is administeredto the subject in accordance with the Physicians' Desk Reference 2003(Thomson Healthcare; 57^(th) edition (Nov. 1, 2002)).

The mode of administration can vary. Routes of administration includeoral, rectal, transmucosal, intestinal, parenteral; intramuscular,subcutaneous, intradermal, intramedullary, intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, intraocular,inhalation, insufflation, topical, cutaneous, transdermal orintra-arterial.

The present invention provides methods for administering an anti-TMPRSS2antigen-binding protein, e.g., antibody or antigen-binding fragmentthereof (e.g., H1H7017N or H4H7017N), comprising introducing the proteininto the body of a subject. For example, the method comprises piercingthe body of the subject with a needle of a syringe and injecting theantigen-binding protein into the body of the subject, e.g., into thevein, artery, tumor, muscular tissue or subcutis of the subject.

The present invention provides a vessel (e.g., a plastic or glass vial,e.g., with a cap or a chromatography column, hollow bore needle or asyringe cylinder) comprising any of the anti-TMPRSS2 antigen-bindingproteins, e.g., antibodies or antigen-binding fragments thereof (e.g.,H1H7017N or H4H7017N), polypeptides (e.g., an HC, LC, V_(H) or V_(L) ofH1H7017N or H4H7017N) or polynucleotides or vectors set forth herein ora pharmaceutical composition thereof comprising a pharmaceuticallyacceptable carrier.

In an embodiment of the invention, an anti-TMPRSS2 antigen-bindingprotein, e.g., antibody or antigen-binding fragment thereof of thepresent invention (e.g., H1H7017N or H4H7017N), is in association withone or more further therapeutic agents. For example, in an embodiment ofthe invention, the further therapeutic agent is an anti-viral drugand/or a vaccine. As used herein, the term “anti-viral drug” refers toany anti-infective drug or therapy used to treat, prevent, or amelioratea viral infection in a subject. The term “anti-viral drug” includes, butis not limited to a cationic steroid antimicrobial, leupeptin,aprotinin, amantadine, rimantadine, oseltamivir, zanamivir, ribavirin,or interferon-alpha2b. Methods for treating or preventing virus (e.g.,influenza) infection in a subject in need of said treatment orprevention by administering H1H7017N or H4H7017N in association with afurther therapeutic agent are part of the present invention.

For example, in an embodiment of the invention, the further therapeuticagent is a vaccine, e.g., an influenza vaccine. In an embodiment of theinvention, a vaccine is an inactivated/killed virus vaccine, a liveattenuated virus vaccine or a virus subunit vaccine.

For example, in an embodiment of the invention, the further therapeuticagent is:

See Shen et al. Biochimie 142: 1-10 (2017).

In an embodiment of the invention, the anti-viral drug is an antibody orantigen-binding fragment that binds specifically to influenza virus,e.g., influenza HA. For example, in an embodiment of the invention, theanti-HA antibody is any one of H1H14611N2; H1H14612N2; H1H11723P;H1H11729P; H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N;H1H11830N2; H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H11711P;H1H11714P; H1H11717P; H1H11724P; H1H11727P; H1H11730P2; H1H11731P2;H1H11734P2; H1H11736P2; H1H11742P2; H1H11744P2; H1H11745P2; H1H11747P2;H1H11748P2; H1H17952B; H1H17953B; H1H17954B; H1H17955B; H1H17956B;H1H17957B; H1H17958B; H1H17959B; H1H17960B; H1H17961B; H1H17962B;H1H17963B; H1H17964B; H1H17965B; H1H17966B; H1H17967B; H1H17968B;H1H17969B; H1H17970B; H1H17971B; H1H17972B; H1H17973B; H1H17974B;H1H17975B; H1H17976B; H1H17977B; H1H17978B; H1H17979B; H1H17980B;H1H17981B; H1H17982B; H1H17983B; H1H17984B; H1H17985B; H1H17986B;H1H17987B; H1H17988B; H1H17989B; H1H17990B; H1H17991B; H1H17992B;H1H17993B; H1H17994B; H1H17995B; H1H17996B; H1H17997B; H1H17998B;H1H17999B; H1H18000B; H1H18001B; H1H18002B; H1H18003B; H1H18004B;H1H18005B; H1H18006B; H1H18007B; H1H18008B; H1H18009B; H1H18010B;H1H18011B; H1H18012B; H1H18013B; H1H18014B; H1H18015B; H1H18016B;H1H18017B; H1H18018B; H1H18019B; H1H18020B; H1H18021B; H1H18022B;H1H18023B; H1H18024B; H1H18025B; H1H18026B; H1H18027B; H1H18028B;H1H18029B; H1H18030B; H1H18031B; H1H18032B; H1H18033B; H1H18034B;H1H18035B; H1H18037B; H1H18038B; H1H18039B; H1H18040B; H1H18041B;H1H18042B; H1H18043B; H1H18044B; H1H18045B; H1H18046B; H1H18047B;H1H18048B; H1H18049B; H1H18051B; H1H18052B; H1H18053B; H1H18054B;H1H18055B; H1H18056B; H1H18057B; H1H18058B; H1H18059B; H1H18060B;H1H18061B; H1H18062B; H1H18063B; H1H18064B; H1H18065B; H1H18066B;H1H18067B; H1H18068B; H1H18069B; H1H18070B; H1H18071B; H1H18072B;H1H18073B; H1H18074B; H1H18075B; H1H18076B; H1H18077B; H1H18078B;H1H18079B; H1H18080B; H1H18081B; H1H18082B; H1H18083B; H1H18084B;H1H18085B; H1H18086B; H1H18087B; H1H18088B; H1H18089B; H1H18090B;H1H18091B; H1H18092B; H1H18093B; H1H18094B; H1H18095B; H1H18096B;H1H18097B; H1H18098B; H1H18099B; H1H18100B; H1H18101B; H1H18102B;H1H18103B; H1H18104B; H1H18105B; H1H18107B; H1H18108B; H1H18109B;H1H18110B; H1H18111B; H1H18112B; H1H18113B; H1H18114B; H1H18115B;H1H18116B; H1H18117B; H1H18118B; H1H18119B; H1H18120B; H1H18121B;H1H18122B; H1H18123B; H1H18124B; H1H18125B; H1H18126B; H1H18127B;H1H18128B; H1H18129B; H1H18130B; H1H18131B; H1H18132B; H1H18133B;H1H18134B; H1H18135B; H1H18136B; H1H18137B; H1H18138B; H1H18139B;H1H18140B; H1H18141B; H1H18142B; H1H18143B; H1H18144B; H1H18145B;H1H18146B; H1H18147B; H1H18148B; H1H18149B; H1H18150B; H1H18151B;H1H18152B; H1H18153B; H1H18154B; H1H18155B; H1H18156B; H1H18157B;H1H18158B; H1H18159B; H1H18160B; H1H18161B; H1H18162B; H1H18163B;H1H18164B; H1H18165B; H1H18166B; H1H18167B; H1H18168B; H1H18169B;H1H18170B; H1H18171B; H1H18172B; H1H18173B; H1H18174B; H1H18175B;H1H18176B; H1H18177B; H1H18178B; H1H18179B; H1H18180B; H1H18181B;H1H18182B; H1H18183B; H1H18184B; H1H18185B; H1H18186B; H1H18187B;H1H18188B; H1H18189B; H1H18190B; H1H18191B; H1H18192B; H1H18193B;H1H18194B; H1H18195B; H1H18196B; H1H18197B; H1H18198B; H1H18199B;H1H18200B; H1H18201B; H1H18202B; H1H18203B; H1H18204B; H1H18205B;H1H18206B; H1H18207B; H1H18208B; H1H18209B; H1H18210B; H1H18211B;H1H18212B; H1H18213B; H1H18214B; H1H18216B; H1H18217B; H1H18218B;H1H18219B; H1H18220B; H1H18221B; H1H18222B; H1H18223B; H1H18224B;H1H18225B; H1H18226B; H1H18227B; H1H18228B; H1H18229B; H1H18230B;H1H18231B; H1H18232B; H1H18233B; H1H18234B; H1H18235B; H1H18236B;H1H18237B; H1H18238B; H1H18239B; H1H18240B; H1H18241B; H1H18242B;H1H18243B; H1H18244B; H1H18245B; H1H18246B; H1H18247B; H1H18248B;H1H18249B; H1H18250B; H1H18251B; H1H18252B; H1H18253B; H1H18254B;H1H18255B; H1H18256B; H1H18257B; H1H18258B; H1H18259B; H1H18261B;H1H18262B; H1H18263B; H1H18264B; H1H18265B; H1H18266B; H1H18267B;H1H18268B; H1H18269B; H1H18270B; H1H18271B; H1H18272B; H1H18274B;H1H18275B; H1H18276B; H1H18277B; H1H18278B; H1H18279B; H1H18280B;H1H18281B; H1H18282B; H1H18283B; H1H18284B; H1H18285B; H1H18286B;H1H18287B; H1H18288B; H1H18289B; H1H18290B; H1H18291B; H1H18292B;H1H18293B; H1H18294B; H1H18295B; H1H18297B; H1H18298B; H1H18299B;H1H18300B; H1H18301B; H1H18302B; H1H18303B; H1H18304B; H1H18305B;H1H18306B; H1H18307B; H1H18308B; H1H18309B; H1H18310B; H1H18311B;H1H18312B; H1H18313B; H1H18314B; H1H18315B; H1H18316B; H1H18317B;H1H18318B; H1H18319B; H1H18320B; H1H18321B; H1H18322B; H1H18323B;H1H18324B; H1H18325B; H1H18326B; H1H18327B; H1H18328B; H1H18329B;H1H18330B; H1H18331B; H1H18332B; H1H18333B; H1H18334B; or H1H18335B; asset forth in International patent application publication no.WO2016/100807; or an antigen-binding fragment thereof, e.g., wherein theantibody or fragment comprises a light chain immunoglobulin thatincludes CDR-L1, CDR-L2 and CDR-L3 (e.g., the V_(L) or light chainthereof); and a heavy chain that includes CDR-H1, CDR-H2 and CDR-H3(e.g., the V_(H) or heavy chain thereof) of any of the foregoinganti-influenza HA antibodies.

In an embodiment of the invention, a further therapeutic agent is anantibody or antigen-binding fragment that binds to influenza Group II HAprotein such as H1H14611N2; or an antibody or fragment that comprisesV_(H) and V_(L) of H1H14611N2; or a heavy chain immunoglobulincomprising CDR-H1, CDR-H2 and CDR-H3 of H1H14611N2 (e.g., SEQ ID NOs:25-27) and a light chain immunoglobulin comprising CDR-L1, CDR-L2 andCDR-L3 of H1H14611N2 (e.g., SEQ ID NOs: 29-31). “H1H14611N2” refers toany anti-group II HA antibody comprising such sequences.

H1H14611N2 Heavy chain variable region (SEQ ID NO: 24)EVQLVESGGGLVKPGGSLRLSCAASGFTFSGFSMNWVRQVPGKGLEWVSSISTSGNYMYYADSVKGRFTISRDNAKKSFSLQMNSLRAEDSAIYYCARGG GYNWNLFDYWGQGSL VTVSSCDR-H1: (SEQ ID NO: 25) GFTFSGFS CDR-H2: (SEQ ID NO: 26) ISTSGNYMCDR-H3: (SEQ ID NO: 27) ARGGGYNWNLFDY Light chain variable region(SEQ ID NO: 28) EIVLTQSPGTLSLSPGERATLSCRASQSLNSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTITRLESEDFAVYYCQQYGNSPLTFG GGTKVEIK CDR-L1:(SEQ ID NO: 29) QSLNSNY CDR-L2: (SEQ ID NO: 30) GAS CDR-L3:(SEQ ID NO: 31) QQYGNSPLT

In an embodiment of the invention, a further therapeutic agent is anantibody or antigen-binding fragment that binds to influenza Group II HAprotein such as H1H14612N2; or an antibody or fragment that comprisesV_(H) and V_(L) of H1H14612N2; or a heavy chain immunoglobulincomprising CDR-H1, CDR-H2 and CDR-H3 of H1H14612N2 (e.g., SEQ ID NOs:41-43) and a light chain immunoglobulin comprising CDR-L1, CDR-L2 andCDR-L3 of H1H14612N2 (e.g., SEQ ID NOs: 45-47). “H1H14612N2” refers toany anti-group II HA antibody comprising such sequences.

H1H14612N2 Heavy chain variable regionEVQLVESGGGLVKPGGSLRLSCAASGFSFSGFSMNWVRQAPGKGLEWVSS ISTSGNYMYY(SEQ ID NO: 40) ADSVKGRFTISRDNAKKSFSLQMNSLRAEDSAIYYCARGGGYNWNLFDYWGQGSLVTVSS CDR-H1: (SEQ ID NO: 41) GFSFSGFS CDR-H2: (SEQ ID NO: 42)ISTSGNYM CDR-H3: (SEQ ID NO: 43) ARGGGYNWNLFDYLight chain variable region (SEQ ID NO: 44)EIVLTQSPGTLSLSPGERATLSCRASQSLNSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGADFTLTISRLESEDFAVYYCQQYGNSPLTFG GGTKVEIK CDR-L1:(SEQ ID NO: 45) QSLNSNY CDR-L2: (SEQ ID NO: 46) GAS CDR-L3:(SEQ ID NO: 47) QQYGNSPLT

In an embodiment of the invention, a further therapeutic agent is anantibody or antigen-binding fragment that binds to influenza Group I HAprotein such as H1H11729P; or an antibody or fragment that comprisesV_(H) and V_(L) of H1H11729P; or a heavy chain immunoglobulin comprisingCDR-H1, CDR-H2 and CDR-H3 of H1H11729P (e.g., SEQ ID NOs: 33-35) and alight chain immunoglobulin comprising CDR-L1, CDR-L2 and CDR-L3 ofH1H11729P (e.g., SEQ ID NOs: 37-39). “H1H11729P” refers to anyanti-group I HA antibody comprising such sequences.

H1H11729P Heavy chain variable region (SEQ ID NO: 32)QVQLVQSGAEVKKSGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTPSYAQKFQDRVTITTDESTSTVYMELSSLRSEDTAVYYCARQQ PVYQYNMDVWGQGTTVTVSSCDR-H1: (SEQ ID NO: 33) GGTFSSYA CDR-H2: (SEQ ID NO: 34) IIPIFGTPCDR-H3: (SEQ ID NO: 35) ARQQPVYQYNMDV Light chain variable region(SEQ ID NO: 36) DIQMTQSPSSLSASVGDRVTITCRASQGIRNNLGWYQQKPLKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYNNYPWTFGQ GTKVEIK CDR-L1:(SEQ ID NO: 37) QURNN CDR-L2: (SEQ ID NO: 38) AAS CDR-L3:(SEQ ID NO: 39) LQYNNYPWT

In a certain embodiment of the invention, the further therapeutic agentis not amantadine, rimantadine, oseltamivir, zanamivir, aprotinin,leupeptin, a cationic steroid antimicrobial, an influenza vaccine (e.g.,killed, live, attenuated whole virus or subunit vaccine), or an antibodyagainst influenza virus (e.g., an anti-hemagglutinin antibody).

The term “in association with” indicates that the components, ananti-TMPRSS2 antigen-binding protein, e.g., antibody or antigen-bindingfragment thereof of the present invention, along with another agent suchas oseltamivir, can be formulated into a single composition, e.g., forsimultaneous delivery, or formulated separately into two or morecompositions (e.g., a kit). Each component can be administered to asubject at a different time than when the other component isadministered; for example, each administration may be givennon-simultaneously (e.g., separately or sequentially) at intervals overa given period of time. Moreover, the separate components may beadministered to a subject by the same or by a different route (e.g.,wherein an anti-TMPRSS2 antibody or antigen-binding fragment thereof.

Kits

Further provided are kits comprising one or more components thatinclude, but are not limited to, an anti-TMPRSS2 antigen-bindingprotein, e.g., an antibody or antigen-binding fragment as discussedherein (e.g., H1H7017N or H4H7017N), in association with one or moreadditional components including, but not limited to, a furthertherapeutic agent, as discussed herein. The antigen-binding proteinand/or the further therapeutic agent can be formulated as a singlecomposition or separately in two or more compositions, e.g., with apharmaceutically acceptable carrier, in a pharmaceutical composition.

In one embodiment of the invention, the kit includes an anti-TMPRSS2antigen-binding protein, e.g., an antibody or antigen-binding fragmentthereof of the invention (e.g., H1H7017N or H4H7017N), or apharmaceutical composition thereof in one container (e.g., in a sterileglass or plastic vial) and a further therapeutic agent in anothercontainer (e.g., in a sterile glass or plastic vial).

In another embodiment, the kit comprises a combination of the invention,including an anti-TMPRSS2 antigen-binding protein, e.g., antibody orantigen-binding fragment thereof of the invention (e.g., H1H7017N orH4H7017N), or pharmaceutical composition thereof in combination with oneor more further therapeutic agents formulated together, optionally, in apharmaceutical composition, in a single, common container.

If the kit includes a pharmaceutical composition for parenteraladministration to a subject, the kit can include a device (e.g., aninjection device) for performing such administration. For example, thekit can include one or more hypodermic needles or other injectiondevices as discussed above containing the anti-TMPRSS2 antigen-bindingprotein, e.g., antibody or antigen-binding fragment thereof of thepresent invention (e.g., H1H7017N or H4H7017N).

The kit can include a package insert including information concerningthe pharmaceutical compositions and dosage forms in the kit. Generally,such information aids patients and physicians in using the enclosedpharmaceutical compositions and dosage forms effectively and safely. Forexample, the following information regarding a combination of theinvention may be supplied in the insert: pharmacokinetics,pharmacodynamics, clinical studies, efficacy parameters, indications andusage, contraindications, warnings, precautions, adverse reactions,overdosage, proper dosage and administration, how supplied, properstorage conditions, references, manufacturer/distributor information andpatent information.

Diagnostic Uses of the Antibodies

The anti-TMPRSS2 antigen-binding proteins, e.g., antibodies orantigen-binding fragments thereof of the present invention (e.g.,H1H7017N or H4H7017N), may be used to detect and/or measure TMPRSS2 in asample. Exemplary assays for TMPRSS2 may include, e.g., contacting asample with an anti-TMPRSS2 antigen-binding protein of the invention,wherein the anti-TMPRSS2 antigen-binding protein is labeled with adetectable label or reporter molecule or used as a capture ligand toselectively isolate TMPRSS2 from samples. The presence of ananti-TMPRSS2 antigen-binding protein complexed with TMPRSS2 indicatesthe presence of TMRPSS2 in the sample. Alternatively, an unlabeledanti-TMPRSS2 antibody can be used in combination with a secondaryantibody which is itself detectably labeled. The detectable label orreporter molecule can be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or¹²⁵I; a fluorescent or chemiluminescent moiety such as fluoresceinisothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase,β-galactosidase, horseradish peroxidase, or luciferase. Specificexemplary assays that can be used to detect or measure TMPRSS2 in asample include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).Thus, the present invention includes a method for detecting the presenceof TMPRSS2 polypeptide in a sample comprising contacting the sample withan anti-TMPRSS2 antigen-binding protein and detecting the presence of aTMPRSS/anti-TMPRSS2 antigen-binding protein wherein the presence of thecomplex indicates the presence of TMPRSS2.

The present invention includes cell-based ELISA methods using theanti-TMPRSS2 antigen-binding proteins, e.g., antibodies andantigen-binding fragments thereof of the present invention (e.g.,H1H7017N), to detect the presence of TMPRSS2 on a cell. In an embodimentof the invention, the method includes the steps:

(i) contacting cells immobilized to a solid surface (e.g., a microplate)to be tested for the presence of TMPRSS2 with an anti-TMPRSS2antigen-binding protein of the present invention;

(ii) optionally washing the mixture to remove unbound anti-TMPRSS2antigen-binding protein;

(iii) contacting the anti-TMPRSS2 antigen-binding protein with a labeledsecondary antibody or antigen-binding fragment thereof that binds to theanti-TMPRSS2 antigen-binding protein;

(iv) optionally washing the complex to remove unbound antigen-bindingprotein; and

(v) detecting the presence of the label on the secondary antibody orfragment, wherein detection of the label indicates that the cellscontain TMPRSS2. For example, the present invention includes suchcell-based ELISA methods for identifying TMPRSS2⁺ cells in a sample.

An anti-TMPRSS2 antigen-binding protein of the invention (e.g., H1H7017Nor H4H7017N) may be used in a Western blot or immune-protein blotprocedure for detecting the presence of TMPRSS2 or a fragment thereof ina sample. Such a procedure forms part of the present invention andincludes the steps of e.g.:

(1) providing a membrane or other solid substrate comprising a sample tobe tested for the presence of TMPRSS2, e.g., optionally including thestep of transferring proteins from a sample to be tested for thepresence of TMPRSS2 (e.g., from a PAGE or SDS-PAGE electrophoreticseparation of the proteins in the sample) onto a membrane or other solidsubstrate using a method known in the art (e.g., semi-dry blotting ortank blotting); and contacting the membrane or other solid substrate tobe tested for the presence of TMPRSS2 or a fragment thereof with ananti-TMPRSS2 antigen-binding protein of the invention.

Such a membrane may take the form, for example, of a nitrocellulose orvinyl-based (e.g., polyvinylidene fluoride (PVDF)) membrane to which theproteins to be tested for the presence of TMPRSS2 in a non-denaturingPAGE (polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodiumdodecyl sulfate polyacrylamide gel electrophoresis) gel have beentransferred (e.g., following electrophoretic separation in the gel).Before contacting the membrane with the anti-TMPRSS2 antigen-bindingprotein, the membrane is optionally blocked, e.g., with non-fat dry milkor the like so as to bind non-specific protein binding sites on themembrane.

(2) washing the membrane one or more times to remove unboundanti-TMPRSS2 antigen-binding protein and other unbound substances; and

(3) detecting the bound anti-TMPRSS2 antigen-binding protein.

Detection of the bound antigen-binding protein indicates that theTMPRSS2 protein is present on the membrane or substrate and in thesample. Detection of the bound antigen-binding protein may be by bindingthe antigen-binding protein with a secondary antibody (ananti-immunoglobulin antibody) which is detectably labeled and, then,detecting the presence of the secondary antibody label.

The anti-TMPRSS2 antigen-binding proteins (e.g., antibodies andantigen-binding fragments (e.g., H1H7017N or H4H7017N)) disclosed hereinmay also be used for immunohistochemistry. Such a method forms part ofthe present invention and comprises, e.g.,

(1) contacting tissue to be tested for the presence of TMPRSS2 proteinwith an anti-TMPRSS2 antigen-binding protein of the invention; and

-   -   (2) detecting the antigen-binding protein on or in the tissue.

If the antigen-binding protein itself is detectably labeled, it can bedetected directly. Alternatively, the antigen-binding protein may bebound by a detectably labeled secondary antibody wherein the label isthen detected.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, room temperatureis about 25° C., and pressure is at or near atmospheric.

Example 1: In Vitro Multicycle Replication

The ability of the influenza virus, A/Puerto Rico/08/1934 (H1N1)-GFP, toreplicate in Calu3, A549, MDCK and HepG2 cells was assessed.

TABLE 1 Reagents used. Description Vendor Calu-3 cells American TypeCulture Collection (ATCC) A549 cells American Type Culture Collection(ATCC) MDCK (London) cells IRR HepG2 cells American Type CultureCollection (ATCC) A/Puerto Rico/08/1934 (H1N1)-GFP N/A DMEM Gibco F12Gibco Pen/Strep Gibco Low IgG BSA Sigma PBS Life Technologies FetalBovine Serum Life TechnologiesExperimental Procedure

Calu-3 cells (ATCC HTB55), A549 cells (ATCC CCL-185), MDCK cells (IRRFR-58) and HepG2 cells (ATCC HB-8065) were diluted to 40,000 cells/wellin a 96-well plate in DMEM:F12 medium with 5% FBS. The next day,A/Puerto Rico/08/1934 (H1N1) carrying a GFP reporter gene in the NSsegment (B. Manicassamy et al., Analysis of in vivo dynamics ofinfluenza virus infection in mice using a GFP reporter virus. Proc NatlAcad Sci USA. 2010 Jun. 22; 107(25):11531-6) was prepared at an MOI(multiplicity of infection) of 0.1 and 0.01 in DMEM:F12 with low IgG BSAafter three washes. The virus was incubated on the cells for 1 h at 37°C. after which the virus was removed and the wells washed three moretimes. The number of infected cells was quantified at 24, 48, 72 and 142h post-infection on a CTL-ImmunoSpot® S6 Universal Analyzer (CellularTechnology Limited, Cleveland, Ohio).

Results Summary and Conclusions

Calu-3 is an immortalized human airway epithelial cell line which hasbeen shown to allow multi-cycle replication of human influenza virusesin the absence of exogenous trypsin (Zeng et al., Highly pathogenicavian influenza H5N1 viruses elicit an attenuated type i interferonresponse in polarized human bronchial epithelial cells. Journal ofVirology. 81, 12439-12449 (2007)). In addition, Calu-3 cells have beenshown to express both TMPRSS2 and TMPRSS4, but not TMPRSS11D (HAT) atleast at the mRNA level (Böttcher-Friebertshäuser et al., Inhibition ofinfluenza virus infection in human airway cell cultures by an antisensepeptide-conjugated morpholino oligomer targeting thehemagglutinin-activating protease TMPRSS2. Journal of Virology. 85,1554-1562 (2011)). To confirm that Calu-3 cells can support theproteolytic activation of influenza virus possessing hemagglutinin witha monobasic cleavage site, the growth of an H1N1 GFP reporter virus inCalu-3 cells was analyzed and replication over time with A549 (humanalveolar basal epithelial), MDCK (Madin Darby canine kidney) and HepG2(human liver carcinoma) cells in the absence of trypsin was compared.The cells were infected at a low MOI and, at the indicated timepoint,viral titers were determined by counting fluorescent focus spots. Table2 and FIG. 1 show low levels of infection in A549, MDCK and HepG2 cells,while Calu-3 cells show significantly increased titers at everytimepoint. Although Calu-3 cells have been shown to express TMPRSS2 andTMPRSS4 at the mRNA level, knockdown of TMPRSS2 reduced influenza virustiters by 100- to 1,000-fold (Böttcher-Friebertshäuser et al.,Inhibition of influenza virus infection in human airway cell cultures byan antisense peptide-conjugated morpholino oligomer targeting thehemagglutinin-activating protease TMPRSS2. Journal of Virology. 85,1554-1562 (2011)). The low level of viral titers in A549, MDCK and HepG2cells in the absence of trypsin are probably due to the addition ofcleaved virus (harvested from embryonated chicken eggs or from MDCKculture with trypsin), but the presence of another HA-activatingprotease could be an explanation.

TABLE 2 Number of infected cells represented by Fluorescent Focus Units(FFU) on different days post-infection with a MOI of 0.1 or 0.01 indifferent cell types after infection with A/Puerto Rico/08/1934(H1N1)-GFP. Cell Day(s) MOI 0.1 MOI 0.01 line post-infection FFU FFUCalu3 1 697 54 2 1167 201 3 1644 376 4 1530 500 A549 1 238 35 2 238 46 3258 53 4 228 52 MDCK 1 740 77 2 750 60 3 879 58 4 796 53 HepG2 1 3 1 214 9 3 20 13 4 21 20

REFERENCES

-   1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates    of Human Coronavirus 229E Bypass the Endosome for Cell Entry.    Journal of Virology. 91, e01387-16 (2017). PMID: 27733646.-   2. L. M. Reinke et al., Different residues in the SARS-CoV spike    protein determine cleavage and activation by the host cell protease    TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 27733646.-   3. Y. Zhou et al., Protease inhibitors targeting coronavirus and    filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID:    25666761.-   4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann,    TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in    Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044.-   5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave    and activate the influenza virus hemagglutinin. PLoS ONE. 12,    e0176597 (2017). PMID: 28493964.-   6. E. Böttcher-Friebertshäuser, D. A. Stein, H.-D. Klenk, W. Garten,    Inhibition of influenza virus infection in human airway cell    cultures by an antisense peptide-conjugated morpholino oligomer    targeting the hemagglutinin-activating protease TMPRSS2. Journal of    Virology. 85, 1554-1562 (2011). PMID: 21123387.-   7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate    trypsin-independent spread of influenza virus in Caco-2 cells.    Journal of Virology. 84, 10016-10025 (2010). PMID: 20631123.-   8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for    pneumotropism and pathogenicity of H7N9 influenza A virus in mice.    Journal of Virology (2014), May;    88(9):4744-51.doi:10.1128/JVI.03799-13. PMID: 24522916.-   9. E. Böttcher et al., Proteolytic Activation of Influenza Viruses    by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium.    Journal of Virology. 2006 October; 80(19):9896-8. PMID: 16973594.-   10. B. Manicassamy et al., Analysis of in vivo dynamics of influenza    virus infection in mice using a GFP reporter virus. Proc Natl Acad    Sci USA. 2010 Jun. 22; 107(25):11531-6. doi:    10.1073/pnas.0914994107. PMID: 20534532.-   11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses    elicit an attenuated type i interferon response in polarized human    bronchial epithelial cells. Journal of Virology. 81, 12439-12449    (2007). PMID: 17855549.

Example 2: Anti-TMPRSS2 Antibody H1H7017N Blocks Spread of Influenza InVitro

The ability of various antibodies to reduce the titers of influenzavirus A/Puerto Rico/08/1934 (H1N1) in Calu-3 cells was assessed.

TABLE 3 Reagents used. Description Vendor Calu-3 cells ATCC F12 GibcoFBS Life Technologies A/Puerto Rico/08/1934 (H1N1) ATCC DMEM GibcoPen/Strep Gibco Low IgG BSA Sigma PBS Life Technologies Paraformaldehyde(16% w/v aq.) Alfa Aesar Triton X-100 EMD Anti-NP antibody MilliporeAnti-Influenza A Antibody, nucleoprotein, clones A1, A3 Blend Goatanti-mouse IgG AF488 conjugated Life TechnologiesExperimental Procedure

Calu-3 cells (ATCC HTB55) were diluted to 40,000 cells/well in a 96-wellplate in DMEM:F12 medium with 5% FBS. The next day, the monoclonalantibodies were diluted to 166.7 nM in DMEM:F12 with low IgG BSA andadded to the cells for 3 h at 37° C. and 5% CO₂. The mAb solution wasremoved and the cells were infected with A/Puerto Rico/08/1934 (H1N1) atan MOI of 0.001. The virus was incubated on the cells for 1 h at 37° C.in 5% CO₂ after which the virus was removed and the medium replaced withDMEM:F12 containing 166.7 nM mAbs. After 24 h and 48 h, the medium wasreplaced with fresh medium containing mAb and the cells were washedtwice with PBS at 72 h. The cells were then fixed with 4%paraformaldehyde in PBS and virus detected using the anti-NP primaryantibody at a 1:1000 dilution. The cells were incubated for 1 h and thenwashed and the secondary at 1:2000 dilution was added. The number ofinfected cells was quantified at on a CTL-ImmunoSpot® S6 UniversalAnalyzer (Cellular Technology Limited, Cleveland, Ohio).

Results Summary and Conclusions

Calu-3 is an immortalized human airway epithelial cell line which hasbeen shown to allow multicycle replication of human influenza viruses inthe absence of exogenous trypsin (Zeng et al., Highly pathogenic avianinfluenza H5N1 viruses elicit an attenuated type i interferon responsein polarized human bronchial epithelial cells. Journal of Virology. 2007November; 81(22):12439-49). In addition, Calu-3 cells have been shown toexpress both TMPRSS2 and TMPRSS4, but not TMPRSS11D (HAT) at least atthe mRNA level (Böttcher-Friebertshäuser et al., Inhibition of influenzavirus infection in human airway cell cultures by an antisensepeptide-conjugated morpholino oligomer targeting thehemagglutinin-activating protease TMPRSS2. Journal of Virology. 85,1554-1562 (2011)). It has been previously shown that Calu-3 cellssupported the proteolytic activation of influenza virus—but inhibitionof TMPRSS2 using the TMPRSS2-specific monoclonal antibody, H1H7017N wastested herein. The growth of A/Puerto Rico/08/1934 (H1N1) over 72 hafter treating the cells with 166.7 nM of H1H7017N was analyzed. Viraltiters were determined by counting fluorescent focus spots. Table 4 andFIG. 2 show decreased titers after treatment with antibody H1H7017N.Although Calu-3 cells have been shown to express TMPRSS2 and TMPRSS4 atthe mRNA level, knockdown of TMPRSS2 reduced influenza virus titers by100- to 1,000-fold (Böttcher-Friebertshäuser et al., Inhibition ofinfluenza virus infection in human airway cell cultures by an antisensepeptide-conjugated morpholino oligomer targeting thehemagglutinin-activating protease TMPRSS2. Journal of Virology. 85,1554-1562 (2011)). The low level of existing viral titers in the absenceof mAb were probably due to the addition of cleaved virus (harvestedfrom embryonated chicken eggs or from MDCK culture with trypsin), butthe presence of another HA-activating protease could also account forthe presence of virus despite treatment with anti-TMPRSS2 mAb.

TABLE 4 Application of H1H7017N during the infection cycle decreases thenumber of Fluorescent Focus Units (FFU) of A/Puerto Rico/08/1934 (H1N1)at 72 hours post-infection. Treatment Description FFU H1H7017NAnti-TMPRSS2 mAb 259 H1H11729P Anti-influenza A group 18 1 positivecontrol anti-hIgG4 with IgG1 isotype control 2338 a mouse IgG2a Fc NomAb Infection control 2656 Uninfected Background control 6

REFERENCES

-   1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates    of Human Coronavirus 229E Bypass the Endosome for Cell Entry.    Journal of Virology. 91, e01387-16 (2017). PMID: 27733646.-   2. L. M. Reinke et al., Different residues in the SARS-CoV spike    protein determine cleavage and activation by the host cell protease    TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 27733646.-   3. Y. Zhou et al., Protease inhibitors targeting coronavirus and    filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID:    25666761.-   4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann,    TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in    Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044.-   5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave    and activate the influenza virus hemagglutinin. PLoS ONE. 12,    e0176597 (2017). PMID: 28493964.-   6. E. Böttcher-Friebertshäuser, D. A. Stein, H.-D. Klenk, W. Garten,    Inhibition of influenza virus infection in human airway cell    cultures by an antisense peptide-conjugated morpholino oligomer    targeting the hemagglutinin-activating protease TMPRSS2. Journal of    Virology. 85, 1554-1562 (2011). PMID: 21123387.-   7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate    trypsin-independent spread of influenza virus in Caco-2 cells.    Journal of Virology. 84, 10016-10025 (2010). PMID: 20631123.-   8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for    pneumotropism and pathogenicity of H7N9 influenza A virus in mice.    Journal of Virology (2014), May;    88(9):4744-51.doi:10.1128/JVI.03799-13. PMID: 24522916.-   9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses    by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium.    Journal of Virology. 2006 October; 80(19):9896-8. PMID: 16973594.-   10. B. Manicassamy et al., Analysis of in vivo dynamics of influenza    virus infection in mice using a GFP reporter virus. Proc Natl Acad    Sci USA. 2010 Jun. 22; 107(25):11531-6. doi:    10.1073/pnas.0914994107. PMID: 20534532.-   11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses    elicit an attenuated type i interferon response in polarized human    bronchial epithelial cells. Journal of Virology. 81, 12439-12449    (2007). PMID: 17855549.

Example 3: FACS Analysis with MDCK/Tet-on, MDCK/Tet-on/hTMPRSS2, andMDCK/Tet-on/MfTMPRSS2 Cells

The ability of anti-TMPRSS2 antibody, H1H7017N, to bind to MDCK cellsexpressing TMPRSS2 or not expressing TMPRSS2 was assessed.

TABLE 5 Reagents used. Reagent Source MDCK ATCC pLVX-EF1α-Tet3G ClontechpLVX Tight hTMPRSS2 Puro pLVX Tight MfTMPRSS2 Puro DMEM IrvineScientific FBS Seradigm Pen/strep/glut Invitrogen Sodium Pyruvate 100 mM(100X) Specialty Media Geneticin ™ Selective Invitrogen Antibiotic (G418Sulfate) Puromycin Sigma Doxycycline Sigma PBS without Ca⁺⁺/Mg⁺⁺ IrvineScientific Accutase Millipore 96-well filter plates Pall BD CytoFix ™Becton Dickinson Allophycocyanin (APC) Jackson Immuno AffiniPure F(ab′)₂Fragment Goat Anti-Human IgG, Fcγ Fragment Specific Control mAb1 (hIgG1isotype control) Cytoflex Beckman Coulter FlowJo 10.1r5 FlowJo Prism 7GraphpadExperimental Procedure

Cell lines were developed to express human and cynomolgous monkeyTMPRSS2 (hTMPRSS2 and mfTMPRSS2) in MDCK (Madin Darby Canine Kidney)cells upon induction with doxycycline. MDCK cells were transduced tostably express a modified tetracycline-controlled transactivator protein(Clontech) and the resulting cell line was termed MDCK/Tet-on cell line.MDCK/Tet-on cell line was transduced with a construct containinghTMPRSS2 (NP_005647.3 with a V160M) or mfTMPRSS2 (Ref seq XP_015302311.1with S129L, N251S, I415V, R431Q, D492G) under the control of induciblepromoter and the cell lines were termed MDCK/Tet-on/hTMPRSS2 andMDCK/Tet-on/mfTMPRSS2. The stable cell lines were maintained in growthmedia containing DMEM supplemented with 10% FBS, sodium pyruvate,penicillin/streptomycin/glutamine, 500 □g/mL G418 with or without 2□g/mL puromycin.

For cell binding analysis by flow cytometry, cells were plated in growthmedia and incubated with doxycycline at 1 □g/mL for 16 hours to induceexpression of TMPRSS2. Cells are detached using Accutase and resuspendedin 1% FBS in PBS. Antibodies were serially diluted from 500 nM to 25 pMand each concentration of antibody was incubated with 1×10⁶ cells at 4°C. for 30 minutes. A condition was included where no antibody was addedto the cells. After incubation with primary antibodies, the cells werestained with allophycocyanin conjugated anti-human IgG secondaryantibody at 1:1000 at 4° C. for 30 minutes. Cells were fixed using BDCytoFix™ and analyzed using CytoFLEX flow cytometer. Unstained andsecondary antibody alone controls were also included for all cell lines.Geometric mean values of fluorescence for viable cells were determinedusing FlowJo software and the results were analyzed using nonlinearregression (4-parameter logistics) with Prism 7 software (GraphPad) toobtain EC₅₀ values of cell binding by the antibodies.

As shown in FIG. 3, the anti-hTMPRSS2 antibody of the invention,H1H7017N, bound to MDCK/Tet-on/hTMPRSS2 and MDCK/Tet-on/mfTMPRSS2 withEC₅₀ values of 460 pM and 1.06 nM respectively. H1H7017N did not showsignificant binding to MDCK/Tet-on cells. Control mAb1, an irrelevantisotype control antibody, did not show binding to any of the cell linestested.

Example 4: Biacore Binding Kinetics of Anti-TMPRSS2 MonoclonalAntibodies Binding to Different TMPRSS2 Reagents Measured at 25° C. and37° C.

Equilibrium dissociation constant (K_(D)) for different TMPRSS2 reagentsbinding to purified anti-TMPRSS2 monoclonal antibodies were determinedusing a real-time surface plasmon resonance based Biacore 4000biosensor. All binding studies were performed in 10 mM HEPES, 150 mMNaCl, 3 mM EDTA, and 0.05% v/v Surfactant Tween-20, pH 7.4 (HBS-ET)running buffer at 25° C. and 37° C. The Biacore CMS sensor chip surfacewas first derivatized by amine coupling with the rabbit anti-mouse Fcspecific polyclonal antibody (GE Healthcare Cat #BR100838) to captureanti-TMPRSS2 monoclonal antibodies. Binding studies were performed onhuman TMPRSS2 extracellular domain expressed with a C-terminalmyc-myc-hexahistidine tag (hTMPRSS2.mmh), and monkey TMPRSS2extracellular domain expressed with a C-terminal myc-myc-hexahistidinetag (mfTMPRSS2.mmh). Different concentrations of HMM-hTMPRSS2 andHMM-mfTMPRSS2 (100 nM-6.25 nM; 4-fold serial dilution) were firstprepared in HBS-ET running buffer and were injected over anti-mouse Fccaptured anti-TMPRSS2 monoclonal antibody surface for 2.5 minutes at aflow rate of 30 μL/minute, while the dissociation of monoclonal antibodybound TMPRSS2 reagent was monitored for 7 minutes in HBS-ET runningbuffer. The association rate (ka) and dissociation rate (kd) weredetermined by fitting the real-time binding sensorgrams to a 1:1 bindingmodel with mass transport limitation using Scrubber 2.0c curve-fittingsoftware. Binding dissociation equilibrium constant (K_(D)) anddissociative half-life (t½) were calculated from the kinetic rates as:

${K_{D} = {(M) = \frac{kd}{ka}}},{{{and}\mspace{14mu}{t^{1/2}\left( \min \right)}} = \frac{\ln(2)}{60*{kd}}}$

Binding kinetics parameters for HMM-hTMPRSS2 or HMM-mfTMPRSS2 binding todifferent anti-TMPRSS2 monoclonal antibodies of the invention at 25° C.and 37° C. are shown in Tables 6 through 9.

At 25° C., anti-TMPRSS2 monoclonal antibodies bound to HMM-hTMPRSS2 withK_(D) value 2.81 nM, as shown in Table 6. At 37° C., anti-HMM-hTMPRSS2monoclonal antibodies bound to HMM-hTMPRSS2 with K_(D) value 9.31 nM, asshown in Table 7.

At 25° C., anti-TMPRSS2 monoclonal antibodies bound to HMM-mfTMPRSS2with K_(D) value 56.0 nM, as shown in Table 8. At 37° C., anti-TMPRSS2monoclonal antibodies bound to HMM-mfTMPRSS2 with K_(D) value 140 nM, asshown in Table 9.

TMPRSS2 Proteins

hTMPRSS2 knob_mmh (W106-R255).mmh:

amino acids 1-150: amino acids 106 through 255 of human TMPRSS2(accession number NP_005647.3 with a V160M)

Amino acids: 151-178: myc-myc-hexahistidine tag

(SEQ ID NO: 20; WKFMGSKCSNSGIECDSSGTCINPSNWCDGVSHCPGGEDENRCVRLYGPNFILQMYSSQRKSWHPVCQDDWNENYGRAACRDMGYKNNFYSSQGIVDDSGSTSFMKLNTSAGNVDIYKKLYHSDACSSKAVVSLRCIACGVNLNSSRQSREQKLISEEDLGGEQKLISEEDL HHHHHH myc tags underscored, His6 tag doubly underscored)

mfTMPRSS2 knob_mmh (W106-R255).mmh:

Amino acids 1-150: amino acids 106-255 of monkey TMPRSS2 (accessionnumber XP_005548700.1 with S129L, N251S)

Amino acids 151-178: myc-myc-hexahistidine tag

(SEQ ID NO: 21; WKFMGSKCSDSGIECDSSGTCISLSNWCDGVSHCPNGEDENRCVRLYGPNFILQVYSSQRKSWHPVCRDDWNENYARAACRDMGYKNSFYSSQGIVDNSGATSFMKLNTSAGNVDIYKKLYHSDACSSKAVVSLRCIACGVRSNLSRQSREQKLISEEDLGGEQKLISEEDL HHHHHH myc tags underscored, His6 tag doubly underscored)Results

TABLE 6 Binding kinetics parameters of HMM-hTMPRSS2 binding to TMPRSS2monoclonal antibodies at 25° C. mAb 100 nM Capture Ag mAb Level Boundk_(a) k_(d) K_(D) t½ Captured (RU) (RU) (1/Ms) (1/s) (M) (min) H2aM7017N510 ± 5.3 103 2.65E+05 7.45E− 2.81E− 15.5 04 09 * H2aM7017N is anantibody with the H1H7017N variable domains set forth herein and a mouseIgG2a Fc.

TABLE 7 Binding kinetics parameters of HMM-hTMPRSS2 binding to TMPRSS2monoclonal antibodies at 37° C. mAb 100 nM Capture Ag mAb Level Boundk_(a) k_(d) K_(D) t½ Captured (RU) (RU) (1/Ms) (1/s) (M) (min) H2aM7017N587 ± 4.5 117 3.47E+05 3.23E− 9.31E− 3.6 03 09

TABLE 8 Binding kinetics parameters of HMM-mfTMPRSS2 binding to TMPRSS2monoclonal antibodies at 25° C. mAb 100 nM Capture Ag mAb Level Boundk_(a) k_(d) K_(D) t½ Captured (RU) (RU) (1/Ms) (1/s) (M) (min) H2aM7017N484 ± 1.8 67 2.80E+05 1.57E− 5.60E− 0.7 02 08

TABLE 9 Binding kinetics parameters of HMM-mfTMPRSS2 binding to MSR1monoclonal antibodies at 37° C. mAb 100 nM Capture Ag mAb Level Boundk_(a) k_(d) K_(D) t½ Captured (RU) (RU) (1/Ms) (1/s) (M) (min) H2aM7017N569 ± 1.6 48 3.66E+05 5.12E− 1.40E− 0.2 02 07

Example 5: In Vitro Influenza Spread of Influenza H1, H3, and FluBStrains

In this example, the ability of various types of influenza to spreadacross an in vitro culture of Calu-3 cells and the effect ofanti-TMPRSS2 antibodies on this spread was determined.

TABLE 10 Reagents used and lot numbers. Cat# Description Vendor HTB55Calu-3 cells ATCC (American Type Culture Collection) 11995-073 DMEMGibco 211703 F12 Gibco 15140-122 Pen/Strep Gibco A033650ML Low IgG BSASigma 10010-023 PBS Life Technologies 26140079 Fetal Bovine Serum LifeTechnologies VR-1469 Influenza A A/Puerto ATCC Rico/08/1934 (H1_PR34)NR-13658 H1N1 A/CaliforniaA/04/2009 BEI Resources (H1_CA09) FR-28Influenza A/Brisbane/59/2007 Influenza Reagent (H1_Bris) ResourceFR-1068 Influenza A/Hong Influenza Reagent Kong/38982/2009 (H9N2)Resource 3483 Influenza A H3N2 Kilbourne BEI Resources F108 A/Aichi/2/68(HA, NA) × A/PR/8/34, Re-assorted X-31 NR-41795 InfluenzaB/Florida/04/2006 ATCC (Florida) NR-12280 Influenza B Malaysia(Malaysia) ATCC MAB8251 Anti-Influenza A Antibody, Milliporenucleoprotein, clones A1, A3 Blend Ab20711 Anti-Influenza B Virus AbcamNucleoprotein antibody [B017] A-11001 Goat anti-Mouse IgG (H + L)ThermoFisher Scientific Cross-Absorbed Secondary Antibody, Alexa Fluor488Experimental Procedure

Calu-3 cells were seeded at 40,000 cells/well in a 96-well plate inDMEM:F12 medium with 5% FBS. The next day, influenza virus strains werediluted to a previously determined MOI (see Table 11) and antibodieswere diluted to 100 μg/mL. In these experiments, the anti-HA andanti-TMPRSS2 antibodies had different mechanisms of action, therefore,the experimental procedure was different for these antibodies in orderto appropriately test them. The anti-HA antibodies were pre-incubatedwith an individual influenza virus strain for one hour at 37° C. in aseparate plate. After the preincubation period, the antibody/virusmixture was added to Calu-3 cells for one hour. The anti-TMPRSS2antibody was preincubated with uninfected Calu-3 cells for three hoursat 37° C. After the preincubation period, virus was added to the Calu-3cells pre-incubated with anti-TMPRSS2 antibodies for one hour. After thehour-long infection, the cells were washed three times with PBS andfresh antibody was, added along with new medium, to each well.Additional antibody was added at 24 and 48 hours post-infection. At 72hours post-infection, the cells were stained with an anti-NP andquantified on a CTL-ImmunoSpot® S6 Universal Analyzer (CellularTechnology Limited, Cleveland, Ohio).

TABLE 11 Influenza Strain Final MOI A. Experiment 1. H1_PR34 0.001H1_CA09 0.001 H1_Bris 0.001 H9N2 0.01 H3N2 0.001 B. Experiment 2.H1_PR34 0.01 Florida 0.01 Malaysia 0.001Results Summary and Conclusions

Calu-3 is an immortalized human airway epithelial cell line which hasbeen shown to allow multicycle replication of human influenza viruses inthe absence of exogenous trypsin (Zeng et al., Journal of Virology 81:12439-12449 (2007)). In addition, Calu-3 cells have been shown toexpress TMPRSS2 (Böttcher-Friebertshäuser et al., Journal of Virology85: 1554-1562 (2011)) which is essential for these experiments as ananti-TMPRSS2 antibody is being tested. In these experiments, whether ornot H1H7017N, an anti-TMPRSS2 antibody, can prevent the spread indifferent strains of influenza was examined. In addition, thecorresponding anti-HA antibody for the different strains as a positivecontrol was run. As expected, there was an initial infection in thepresence of the anti-TMPRSS2 antibody but H1H7017N successfullyprevented the spread of infection of H1_PR34, H1_CA09, H1_Bris, H9N2,and H3N2. This can be observed by examining the differences in thenumber of infected cells between the anti-TMPRSS2-treated cells and theinfected controls (Table 12). It was concluded that the anti-TMPRSS2antibody was not able to prevent spread in either of the influenza Bstrains because the number of infected cells in the control and treatedwells were the same. In comparison, the anti-HA antibodies werepre-incubated with the virus and prevented the initial infection. Thiscan also be seen by comparing the number of infected cells. Counting ofthe infected cells was performed on the CTL machine and are reported inthe tables below.

TABLE 12 Infected cells treated with Infected cells H1H7017N treatedwith anti- Influenza Uninfected Infected group specific TMPRSS2 StrainControl Control HA antibody antibody A. Experiment 1. H1_PR34 10 3847.53 1496 H1_CA09 3.5 4645.4 1.5 17 H1_Bris 15.5 3882 0.5 1005 H9N2 4.54172 4.5 196.5 H3N2 7.5 3922 9 754.5 B. Experiment 2. H1_PR34 1 2848 1860 Florida 4 1339 229 1234 Malaysia 10 1184 758 1451

REFERENCES

-   1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates    of Human Coronavirus 229E Bypass the Endosome for Cell Entry.    Journal of Virology. 91, e01387-16 (2017). PMID: 27733646.-   2. L. M. Reinke et al., Different residues in the SARS-CoV spike    protein determine cleavage and activation by the host cell protease    TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 28636671.-   3. Y. Zhou et al., Protease inhibitors targeting coronavirus and    filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID:    25666761.-   4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann,    TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in    Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044.-   5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave    and activate the influenza virus hemagglutinin. PLoS ONE. 12,    e0176597 (2017). PMID: 28493964.-   6. E. Böttcher-Friebertshäuser, D. A. Stein, H.-D. Klenk, W. Garten,    Inhibition of influenza virus infection in human airway cell    cultures by an antisense peptide-conjugated morpholino oligomer    targeting the hemagglutinin-activating protease TMPRSS2. Journal of    Virology. 85, 1554-1562 (2011). PMID: 21123387.-   7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate    trypsin-independent spread of influenza virus in Caco-2 cells.    Journal of Virology. 84, 10016-10025 (2010). PMID: 20631123.-   8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for    pneumotropism and pathogenicity of H7N9 influenza A virus in mice.    Journal of Virology (2014), doi:10.1128/JVI.03799-13. PMID:    24522916.-   9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses    by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium.    Journal of Virology. 80, 9896-9898 (2006). PMID: 16973594.-   10. Manicassamy et al., Analysis of in vivo dynamics of influenza    virus infection in mice using a GFP reporter virus. Proc Natl Acad    Sci USA. 2010 Jun. 22; 107(25):11531-6. doi:    10.1073/pnas.0914994107. Epub 2010 Jun. 7. PMID: 20534532.-   11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses    elicit an attenuated type i interferon response in polarized human    bronchial epithelial cells. Journal of Virology. 81, 12439-12449    (2007). PMID: 17855549.

Example 6: The Effect of Treatment with H1H7017N Alone in TMPRS22Humanized Mice

The ability of anti-TMPRSS2 antibodies to protect mice engineered toexpress the human TMPRSS2 protein from infection with H1N1 influenzavirus was assessed.

TABLE 13 Reagents used and lot numbers. Cat# Description Vendor VR-1469Influenza A A/Puerto ATCC Rico/08/1934 (H1N1) 20012-043 PBS GibcoKetamine:Xylazine

TABLE 14 mAb Clone IDs. AbPID Description H1H7017N anti-TMPRSS2 mAbH1H1238N IgG1 isotype controlExperimental Procedure

These experiments were performed in 5-8 week-old male and female miceengineered to express the human TMPRSS2 protein. Mice were challengedwith 150 plaque-forming units (PFUs) of H1N1. The mice were sedated with200 μL of Ketamine:Xylazine (12 mg/ml:0.5 mg/ml) via intraperitonealinjection and then infected with 24 μL of virus intranasally. Antibodieswere delivered either subcutaneously (SC) one day before infection orintravenously (IV) on various days post infection (PI). The antibodydosing schedule varied between experiments (Table 15). Body weights werecollected daily up to day 14 PI and mice were sacrificed when they lost20% of their starting body weight. Results are reported as percentsurvival.

TABLE 15 Antibody Days PI Dose Delivery A. Antibody Dosing (Experiment1). H1H1238N −1  5 mg/kg SC H1H7017N −1, 0 5 mg/kg SC, IV B. AntibodyDosing (Experiment 2). H1H1238N 0 10 mg/kg IV H1H7017N 0, 1, 2, 3 10mg/kg IVResults Summary and Conclusions

It has been shown that mice engineered to express the human TMPRSS2protein can be infected with a lethal dose of influenza. The aim ofthese experiments was to demonstrate that H1H7017N can protect miceengineered to express the human TMPRSS2 protein against influenza Agroup 1. The antibody was tested in prophylactic and therapeutic models.Treatment with H1H7017N resulted in higher survival than the isotypecontrol (H1H1238N) treated mice in both experiments (FIGS. 4 and 5). Inthe prophylactic experiment, the survival was 0% for mice treated withH1H1238N, 85.7% for mice treated on day −1 PI, and 100% for mice treatedon day 0 PI with H1H7017N. For the therapeutic model, theH1H1238N-treated group resulted in 25% survival while the groups treatedwith H1H7017N on day 0-3 PI resulted in 100% survival. Data aresummarized in Table 16. H1H7017N shows efficacy in mice engineered toexpress the human TMPRSS2 protein.

TABLE 16 Percent survival Number (no. of surviving of mice mice/totalno. of Group ID per group mice in the group) A. Tabulated Data Summary(Experiment 1). H1H1238N, Day −1 PI, SC 4 0 (0/4) H1H7017N, Day −1 PI,SC 7 85.7 (6/7) H1H7017N, Day 0 PI, IV 6 100 (6/6) B. Tabulated DataSummary (Experiment 2). H1H1238N, Day 0 PI, IV 4 25 (1/4) H1H7017N, Day0 PI, IV 5 100 (5/5) H1H7017N, Day 1 PI, IV 5 100 (5/5) H1H7017N, Day 2PI, IV 5 100 (5/5) H1H7017N, Day 3 PI, IV 5 100 (5/5)

REFERENCES

-   1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates    of Human Coronavirus 229E Bypass the Endosome for Cell Entry.    Journal of Virology. 91, e01387-16 (2017). PMID: 27733646.-   2. L. M. Reinke et al., Different residues in the SARS-CoV spike    protein determine cleavage and activation by the host cell protease    TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 28636671.-   3. Y. Zhou et al., Protease inhibitors targeting coronavirus and    filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID:    25666761.-   4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann,    TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in    Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044.-   5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave    and activate the influenza virus hemagglutinin. PLoS ONE. 12,    e0176597 (2017). PMID: 28493964.-   6. E. Böttcher-Friebertshäuser, D. A. Stein, H.-D. Klenk, W. Garten,    Inhibition of influenza virus infection in human airway cell    cultures by an antisense peptide-conjugated morpholino oligomer    targeting the hemagglutinin-activating protease TMPRSS2. Journal of    Virology. 85, 1554-1562 (2011). PMID: 21123387.-   7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate    trypsin-independent spread of influenza virus in Caco-2 cells.    Journal of Virology. 84, 10016-10025 (2010). PMID: 20631123.-   8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for    pneumotropism and pathogenicity of H7N9 influenza A virus in mice.    Journal of Virology (2014), doi:10.1128/JVI.03799-13. PMID:    24522916.-   9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses    by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium.    Journal of Virology. 80, 9896-9898 (2006). PMID: 16973594.-   10. Manicassamy et al., Analysis of in vivo dynamics of influenza    virus infection in mice using a GFP reporter virus. Proc Natl Acad    Sci USA. 2010 Jun. 22; 107(25):11531-6. doi:    10.1073/pnas.0914994107. Epub 2010 Jun. 7. PMID: 20534532.-   11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses    elicit an attenuated type i interferon response in polarized human    bronchial epithelial cells. Journal of Virology. 81, 12439-12449    (2007). PMID: 17855549.

Example 7: Anti-TMPRSS2 mAb, H1H7017N, Activity in TMPRSS2 HumanizedMouse Model

The ability of anti-TMPRSS2 antibodies to protect a mouse engineered toexpress the human TMPRSS2 protein from infection with H3N2 influenzavirus was assessed.

TABLE 17 mAb Clone IDs. AbPID Description H1H7017N Anti-TMPRSS2 antibody

TABLE 18 Reagents used and lot numbers. Cat# Description Vendor 3483Influenza A H3N2 Kilbourne F108 BEI Resources A/Aichi/2/68 (HA, NA) ×A/PR/8/34, Reassorted X-31 20012-043 PBS Gibco Ketamine:XylazineExperimental Procedure

Eleven week-old male and female mice engineered to express the humanTMPRSS2 protein were challenged with 20,000 plaque-forming units (PFUs)of H3N2. The mice were sedated with 200 μL of Ketamine:Xylazine (12mg/ml:0.5 mg/ml) via intraperitoneal injection and then infected with 20μL of virus intranasally. On day 1 or day 2 post-infection (PI), micewere intravenously injected with antibody. Mice were weighed andobserved daily up to day 14 post-infection (PI). They were sacrificedwhen they lost 25% of their starting body weight.

Results Summary and Conclusions

Breadth is an important quality when considering an influenza therapy.It has already been demonstrated that anti-TMPRSS2 antibody H1H7017N wasefficacious against influenza A group 1. The aim of this experiment wasto demonstrate that H1H7017N can protect mice engineered to express thehuman TMPRSS2 protein against influenza A group 2. Mice engineered toexpress the human TMPRSS2 protein were infected with a lethal dose ofH3N2 and treated on day 1 or day 2 PI. Both treatment groups had highersurvival rates than the infected control. Mice treated on day 1 PI had asurvival rate of 100% which was higher than the group treated on day 2PI which had a 50% survival, while untreated mice had 0% survival. Allmice died between days 5-6 PI. The survival graph is shown in FIG. 6 and% survival is summarized in Table 19. These results demonstrated thatH1H7017 improved outcomes in an H3N2-lethal model.

TABLE 19 Tabulated Data Summary. Percent survival Number (no. ofsurviving of mice mice/total no. of Group ID per group mice in thegroup) Untreated 5 0 (0/5) H1H7017N, Day 1 PI 5 100 (5/5) H1H7017N, Day0 PI, IV 4 50 (2/4)

REFERENCES

-   1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates    of Human Coronavirus 229E Bypass the Endosome for Cell Entry.    Journal of Virology. 91, e01387-16 (2017). PMID: 27733646.-   2. L. M. Reinke et al., Different residues in the SARS-CoV spike    protein determine cleavage and activation by the host cell protease    TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 28636671.-   3. Y. Zhou et al., Protease inhibitors targeting coronavirus and    filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID:    25666761.-   4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann,    TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in    Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044.-   5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave    and activate the influenza virus hemagglutinin. PLoS ONE. 12,    e0176597 (2017). PMID: 28493964.-   6. E. Böttcher-Friebertshäuser, D. A. Stein, H.-D. Klenk, W. Garten,    Inhibition of influenza virus infection in human airway cell    cultures by an antisense peptide-conjugated morpholino oligomer    targeting the hemagglutinin-activating protease TMPRSS2. Journal of    Virology. 85, 1554-1562 (2011). PMID: 21123387.-   7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate    trypsin-independent spread of influenza virus in Caco-2 cells.    Journal of Virology. 84, 10016-10025 (2010). PMID: 20631123.-   8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for    pneumotropism and pathogenicity of H7N9 influenza A virus in mice.    Journal of Virology (2014), doi:10.1128/JVI.03799-13. PMID:    24522916.-   9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses    by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium.    Journal of Virology. 80, 9896-9898 (2006). PMID: 16973594.-   10. Manicassamy et al., Analysis of in vivo dynamics of influenza    virus infection in mice using a GFP reporter virus. Proc Natl Acad    Sci USA. 2010 Jun. 22; 107(25):11531-6. doi:    10.1073/pnas.0914994107. Epub 2010 Jun. 7. PMID: 20534532.-   11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses    elicit an attenuated type i interferon response in polarized human    bronchial epithelial cells. Journal of Virology. 81, 12439-12449    (2007). PMID: 17855549.

Example 8: Infection of Mice Engineered to Express the Human TMPRSS2Protein (Versus WT)

The survival of mice engineered to express the human TMPRSS2 proteininfected with H1N1 influenza virus was assessed and compared with thatof wild-type (WT) mice.

TABLE 20 Reagents used and lot numbers. Cat# Description Vendor VR-1469Influenza A A/Puerto ATCC Rico/08/1934 (H1N1) 20012-043 PBS GibcoKetamine:XylazineExperimental Procedure

The experiment was performed in 7.5-8 week-old male and female miceengineered to express the human TMPRSS2 protein or wild-typelittermates. Mice were challenged with 150, 750, or 1,500 plaque-formingunits (PFUs) of A/Puerto Rico/08/1934 (H1N1). The mice were sedated with200 μL of Ketamine:Xylazine (12 mg/ml:0.5 mg/ml) via intraperitonealinjection and then infected with 20 μL of virus intranasally. Bodyweights were collected daily up to day 14 PI and mice were sacrificedwhen they lost 20% of their starting body weight. Results are reportedas percent survival (FIG. 7).

Results Summary and Conclusions

Mice engineered to express the human TMPRSS2 protein were generated inorder to test the therapeutic efficacy of the anti-TMPRSS2 antibodies inan influenza in vivo model. In this experiment, the survival rates ofmice engineered to express the human TMPRSS2 protein and wild-type miceinfected with 150, 750 or 1,500 PFUs of a historical strain of H1N1 wascompared. There was 0% survival for mice engineered to express the humanTMPRSS2 protein and wild-type mice in all three infection groups. Allmice died between day 5 and day 8 PI, with those receiving a highervirus dose dying sooner than those who received a lower virus dose. Thesurvival patterns of mice engineered to express the human TMPRSS2protein were similar to the wild-type mice. This shows that miceengineered to express the human TMPRSS2 protein can be used as aninfluenza in vivo model to assess the effectiveness of TMPRSS2-specificantibodies. See Table 21.

TABLE 21 Tabulated Data Summary. Percent survival Number (no. ofsurviving of mice mice/total no. of Group ID per group mice in thegroup) Wild-type; 150 PFUs H1N1 4 0 (0/4) Wild-type; 750 PFUs H1N1 4 0(0/4) Wild-type; 1,500 PFUs H1N1 3 0 (0/3) Mice engineered to expressthe human 4 0 (0/4) TMPRSS2 protein; 150 PFUs H1N1 Mice engineered toexpress the human 3 0 (0/3) TMPRSS2 protein; 750 PFUs H1N1 Miceengineered to express the human 3 0 (0/3) TMPRSS2 protein; 1,500 PFUsH1N1

REFERENCES

-   1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates    of Human Coronavirus 229E Bypass the Endosome for Cell Entry.    Journal of Virology. 91, e01387-16 (2017). PMID: 27733646.-   2. L. M. Reinke et al., Different residues in the SARS-CoV spike    protein determine cleavage and activation by the host cell protease    TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 28636671.-   3. Y. Zhou et al., Protease inhibitors targeting coronavirus and    filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID:    25666761.-   4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann,    TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in    Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044.-   5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave    and activate the influenza virus hemagglutinin. PLoS ONE. 12,    e0176597 (2017). PMID: 28493964.-   6. E. Böttcher-Friebertshäuser, D. A. Stein, H.-D. Klenk, W. Garten,    Inhibition of influenza virus infection in human airway cell    cultures by an antisense peptide-conjugated morpholino oligomer    targeting the hemagglutinin-activating protease TMPRSS2. Journal of    Virology. 85, 1554-1562 (2011). PMID: 21123387.-   7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate    trypsin-independent spread of influenza virus in Caco-2 cells.    Journal of Virology. 84, 10016-10025 (2010). PMID: 20631123.-   8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for    pneumotropism and pathogenicity of H7N9 influenza A virus in mice.    Journal of Virology (2014), doi:10.1128/JVI.03799-13. PMID:    24522916.-   9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses    by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium.    Journal of Virology. 80, 9896-9898 (2006). PMID: 16973594.-   10. Manicassamy et al., Analysis of in vivo dynamics of influenza    virus infection in mice using a GFP reporter virus. Proc Natl Acad    Sci USA. 2010 Jun. 22; 107(25):11531-6. doi:    10.1073/pnas.0914994107. Epub 2010 Jun. 7. PMID: 20534532.-   11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses    elicit an attenuated type i interferon response in polarized human    bronchial epithelial cells. Journal of Virology. 81, 12439-12449    (2007). PMID: 17855549.

Example 9: The Effect of Treatment with the Combination of H1H14611N2and H1H7017N in Mice after Infection with H3N2

The ability of a combination of anti-TMPRSS2 and anti-influenzaantibodies to protect mice engineered to express the human TMPRSS2protein from infection with H3N2 influenza virus was assessed.

TABLE 22 mAb Clone IDs. AbPID Description H1H7017N Anti-TMPRSS2 antibodyH1H14611N2 Anti-influenza A group 2 antibody H1H1238N IgG1 isotypecontrol

TABLE 23 Reagents used and lot numbers. Description Vendor Influenza AH3N2 Kilbourne BEI Resources F108 A/Aichi/2/68 (HA, NA) × A/PR/8/34,Reassorted X-31 PBS Gibco Ketamine:XylazineExperimental Procedure

Eight week-old male and female mice engineered to express the humanTMPRSS2 protein were challenged with 20,000 plaque-forming units (PFUs)of A/Aichi/2/68 (HA, NA)×A/PR/8/34, Re*assorted X-31 (H3N2). The micewere sedated with 200 μL of Ketamine:Xylazine (12 mg/ml:0.5 mg/ml) viaintraperitoneal injection and then infected with 20 μL of virusintranasally. On day 4 post-infection (PI), mice were intravenouslyinjected with antibody. Body weights were collected daily up to day 14PI and mice were sacrificed when they lost 25% of their starting bodyweight. Results are reported as percent survival.

Results Summary and Conclusions

It has been shown that, individually, the TMPRSS2 antibody, H1H7017N,and the broad influenza A group 2 antibody, H1H14611N2, have therapeuticefficacy against a lethal mouse challenge with a historical strain ofH3N2. It has also been shown that survival of mice infected with alethal H1N1 challenge can be significantly increased after treatmentwith less total antibody than either alone through the combination ofH1H7017N and the broad influenza A group 1 antibody, H1H11729P. The aimfor this experiment was to evaluate the synergistic effect of H1H7017Nand H1H14611N2 in combination. As shown in FIG. 8, 3 of 4 mice treatedwith the hIgG1 isotype control antibody at day 4 PI died by day 7 PI. 3of 5 animals survived when dosed with 10 mg/kg of H1H14611N2 and 4 of 5animals survived when dosed with 10 mg/kg of H1H7017N. When dosed in acombination of 5 mg/kg of each antibody, H1H14611N2 and H1H7017N, therewas 40% survival. One hundred percent of mice treated with thecombination of 2.5 mg/kg of each antibody, H1H14611N2 and H1H7017N,survived the challenge. Survival of mice infected with a lethal H3N2challenge was increased through the combination of lower concentrationsof H1H7017N and H1H14611N2 compared to higher concentrations of combinedantibodies or either antibody alone. Percent survival is summarized inTable 24.

TABLE 24 Tabulated Data Summary. Percent survival Number (no. ofsurviving of mice mice/total no. of Group ID per group mice in thegroup) 10 mg/kg hIgG1 isotype control 5 20 (1/5) 10 mg/kg H1H14611N2 560 (3/5) 10 mg/kg H1H7017N 5 80 (4/5) 5 mg/kg H1H7017N + 5 mg/kg 5 40(2/5) H1H14611N2 2.5 mg/kg H1H7017N + 2.5 mg/kg 5 100 (5/5) H1H14611N2

REFERENCES

-   1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates    of Human Coronavirus 229E Bypass the Endosome for Cell Entry.    Journal of Virology. 91, e01387-16 (2017). PMID: 27733646.-   2. L. M. Reinke et al., Different residues in the SARS-CoV spike    protein determine cleavage and activation by the host cell protease    TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 28636671.-   3. Y. Zhou et al., Protease inhibitors targeting coronavirus and    filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID:    25666761.-   4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann,    TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in    Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044.-   5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave    and activate the influenza virus hemagglutinin. PLoS ONE. 12,    e0176597 (2017). PMID: 28493964-   6. E. Böttcher-Friebertshäuser, D. A. Stein, H.-D. Klenk, W. Garten,    Inhibition of influenza virus infection in human airway cell    cultures by an antisense peptide-conjugated morpholino oligomer    targeting the hemagglutinin-activating protease TMPRSS2. Journal of    Virology. 85, 1554-1562 (2011). PMID: 21123387.-   7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate    trypsin-independent spread of influenza virus in Caco-2 cells.    Journal of Virology. 84, 10016-10025 (2010). PMID: 20631123.-   8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for    pneumotropism and pathogenicity of H7N9 influenza A virus in mice.    Journal of Virology (2014), doi:10.1128/JVI.03799-13. PMID:    24522916.-   9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses    by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium.    Journal of Virology. 80, 9896-9898 (2006). PMID: 16973594.-   10. Manicassamy et al., Analysis of in vivo dynamics of influenza    virus infection in mice using a GFP reporter virus. Proc Natl Acad    Sci USA. 2010 Jun. 22; 107(25):11531-6. doi:    10.1073/pnas.0914994107. Epub 2010 Jun. 7. PMID: 20534532.-   11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses    elicit an attenuated type i interferon response in polarized human    bronchial epithelial cells. Journal of Virology. 81, 12439-12449    (2007). PMID: 17855549.

Example 10: The Effect of Treatment with the Combination of H1H11729Pand H1H7017N in Mice after Infection with H1N1

The ability of a combination of anti-TMPRSS2 and anti-influenzaantibodies to protect mice engineered to express the human TMPRSS2protein from infection with H1N1 influenza virus was assessed.

TABLE 25 mAb Clone IDs. AbPID Description H1H7017N Anti-TMPRSS2 antibodyH1H11729P Anti-influenza A group 1 antibody H1H1238N IgG1 isotypecontrol

TABLE 26 Reagents used and lot numbers. Cat# Description Vendor VR-1469Influenza A A/Puerto ATCC Rico/08/1934 (H1N1) 20012-043 PBS GibcoKetamine:XylazineExperimental Procedure

Five week-old male and female mice engineered to express the humanTMPRSS2 protein were challenged with 1,500 plaque-forming units (PFUs)of H1N1. The virus was delivered by sedating the mice with 200 μL ofKetamine:Xylazine (12 mg/ml:0.5 mg/ml) and delivering 20 μL of virusintranasally. On day 3 post-infection (PI), mice were intravenouslyinjected with antibody. Body weights were collected daily up to day 14PI and mice were sacrificed when they lost 25% of their starting bodyweight.

Results Summary and Conclusions

It has been shown that, individually, the TMPRSS2 antibody, H1H7017N,and the broad influenza A group 1 antibody, H1H11729P, have therapeuticefficacy against a lethal mouse challenge with a historical strain ofH1N1. However, the aim of this experiment was to evaluate thesynergistic effect of the antibodies in combination. All mice treatedwith hIgG1 isotype control antibody at day 3 PI died by day 6 PI. Whenanimals received 5 mg/kg of H1H11729P or H1H7017N, 40% and 0% of animalssurvived the infection, respectively. However, the combination of 2.5mg/kg of each antibody, H1H11729P and H1H7017N, resulted in 60%survival. Eighty percent of mice treated with the combination of 1 mg/kgof H1H7017N and 2 mg/kg of H1H11729P (3 mg/kg total) survived thechallenge. Survival of mice infected with a lethal H1N1 challenge wassignificantly increased after treatment with less total antibody thaneither alone through the combination H1H7017N and H1H11729P (See FIG. 9and Table 27).

TABLE 27 Tabulated Data Summary. Percent survival Number (no. ofsurviving of mice mice/total no. of Group ID per group mice in thegroup) 5 mg/kg hIgG1 isotype control 3 0 (0/3) 5 mg/kg H1H11729P 5 40(2/5) 5 mg/kg H1H7017N 5 0 (0/5) 2.5 mg/kg H1H7017N + 2.5 mg/kg 5 60(3/5) H1H11729P 1 mg/kg H1H7017N + 2 mg/kg 5 80 (4/5) H1H11729P

REFERENCES

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All references cited herein are incorporated by reference to the sameextent as if each individual publication, database entry (e.g., Genbanksequences or GeneID entries), patent application, or patent, wasspecifically and individually indicated to be incorporated by reference.This statement of incorporation by reference is intended by Applicantsto relate to each and every individual publication, database entry(e.g., Genbank sequences or GeneID entries), patent application, orpatent identified even if such citation is not immediately adjacent to adedicated statement of incorporation by reference. The inclusion ofdedicated statements of incorporation by reference, if any, within thespecification does not in any way weaken this general statement ofincorporation by reference. Citation of the references herein is notintended as an admission that the reference is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

I claim:
 1. A human antigen-binding protein that specifically binds tohuman TMPRSS2, comprising: (a) an immunoglobulin heavy chain variableregion comprising the CDR-H1, CDR-H2, and CDR-H3 of an immunoglobulinheavy chain that comprises the amino acid sequence set forth in SEQ IDNO: 2, 17 or 19; and/or (b) an immunoglobulin light chain variableregion comprising the CDR-L1, CDR-L2, and CDR-L3 of an immunoglobulinlight chain that comprises the amino acid sequence set forth in SEQ IDNO: 4 or
 18. 2. The antigen-binding protein of claim 1 which is anantibody or antigen-binding fragment thereof.
 3. A human antigen-bindingprotein that specifically binds to human TMPRSS2, comprising: a lightchain immunoglobulin variable region that comprises: (a) a CDR-L1comprising the amino acid sequence: QSISSW (SEQ ID NO: 12), (b) a CDR-L2comprising the amino acid sequence: K A S (SEQ ID NO: 14), (c) a CDR-L3comprising the amino acid sequence: QQYNSYSYT (SEQ ID NO: 16); and aheavy chain immunoglobulin variable region that comprises: (a) a CDR-H1comprising the amino acid sequence: GFTFSSYG (SEQ ID NO: 6), (b) aCDR-H2 comprising the amino acid sequence: IWNDGSYV (SEQ ID NO: 8), (c)a CDR-H3 comprising the amino acid sequence: AREGEWVLYYFD Y (SEQ ID NO:10).
 4. A human antigen-binding protein that specifically binds to humanTMPRSS2, comprising: (a) a heavy chain immunoglobulin that comprises theamino acid sequence set forth in SEQ ID NO: 17 or 19, or animmunoglobulin heavy chain variable region that comprises the amino acidsequence set forth in SEQ ID NO: 2; and/or (b) a light chainimmunoglobulin that comprises the amino acid sequence set forth in SEQID NO: 18, or an immunoglobulin light chain variable region thatcomprises the amino acid sequence set forth in SEQ ID NO:
 4. 5. Anantigen-binding protein that competes with an antigen-binding protein ofclaim 1 for binding to TMPRSS2 and/or binds to the same or anoverlapping epitope on TMPRSS2.
 6. The antigen-binding protein of claim1 which is multispecific.
 7. The antigen-binding protein of claim 1which comprises one or more of the following properties: Inhibits growthof influenza virus in TMPRSS2-expressing cells; Binds to the surface ofTMPRSS-expressing cells; Does not significantly bind to MDCK/Tet-oncells which do not express TMPRSS2; Binds to human TMPRSS2 with a K_(D)of about 2.81×10⁻⁹M at about 25° C.; Binds to human TMPRSS2 with a K_(D)of about 9.31×10⁻⁹M at about 37° C.; Binds to cynomolgus TMPRSS2 with aK_(D) of about 5.60×10⁻⁸M at about 25° C.; Binds to cynomolgus TMPRSS2with a K_(D) of about 1.40×10⁻⁷M at about 37° C.; Limits spread ofinfluenza virus infection of cells in vitro; and/or Protects miceengineered to express the human TMPRSS2 protein from death caused byinfluenza virus infection.
 8. A complex comprising an antigen-bindingprotein of claim 1 bound to a TMPRSS2 polypeptide.
 9. A method formaking an antigen-binding protein of claim 1 or immunoglobulin chainthereof comprising: (a) introducing one or more polynucleotides encodingan immunoglobulin chain of said antigen-binding protein; (b) culturingthe host cell under conditions favorable to expression of thepolynucleotide; and (c) optionally, isolating the antigen-bindingprotein or immunoglobulin chain from the host cell and/or medium inwhich the host cell is grown.
 10. The method of claim 9 wherein the hostcell is a Chinese hamster ovary cell.
 11. A polypeptide comprising: (a)CDR1, CDR2, and CDR3 of a V_(H) domain of an immunoglobulin chain thatcomprises the amino acid sequence set forth in SEQ ID NO: 2; or (b)CDR1, CDR2, and CDR3 of a V_(L) domain of an immunoglobulin chain thatcomprises the amino acid sequence set forth in SEQ ID NO:
 4. 12. Apolynucleotide encoding the polypeptide of claim
 11. 13. A vectorcomprising the polynucleotide of claim
 12. 14. A host cell comprisingthe vector of claim
 12. 15. A composition or kit comprising theantigen-binding protein of claim 1 in association with a furthertherapeutic agent.
 16. A pharmaceutical composition comprising theantigen-binding protein of claim 1 and pharmaceutically acceptablecarrier and, optionally, a further therapeutic agent.
 17. Thecomposition or kit of claim 15 in association with a further therapeuticagent which is an anti-viral drug or a vaccine.
 18. The composition orkit of claim 17 wherein the further therapeutic agent is a memberselected from the group consisting of: ledipasvir, sofosbuvir, acombination of ledipasvir and sofosbuvir, oseltamivir, zanamivir,ribavirin and interferon-alpha2b, interferon-alpha2a, an anti-canceragent and an antibody or antigen-binding fragment thereof thatspecifically binds to influenza HA; and/or an antibody or antigenbinding fragment thereof selected from the group consisting ofH1H14611N2; H1H14612N2; H1H11723P; H1H11729P; H1H11820N; H1H11829N;H1H11829N2; H2aM11829N; H2M11830N; H1H11830N2; H1H11903N; H1H14571N;H2a14571N; H1H11704P; H1H11711P; H1H11714P; H1H11717P; H1H11724P;H1H11727P; H1H11730P2; H1H11731P2; H1H11734P2; H1H11736P2; H1H11742P2;H1H11744P2; H1H11745P2; H1H11747P2; H1H11748P2; H1H17952B; H1H17953B;H1H17954B; H1H17955B; H1H17956B; H1H17957B; H1H17958B; H1H17959B;H1H17960B; H1H17961B; H1H17962B; H1H17963B; H1H17964B; H1H17965B;H1H17966B; H1H17967B; H1H17968B; H1H17969B; H1H17970B; H1H17971B;H1H17972B; H1H17973B; H1H17974B; H1H17975B; H1H17976B; H1H17977B;H1H17978B; H1H17979B; H1H17980B; H1H17981B; H1H17982B; H1H17983B;H1H17984B; H1H17985B; H1H17986B; H1H17987B; H1H17988B; H1H17989B;H1H17990B; H1H17991B; H1H17992B; H1H17993B; H1H17994B; H1H17995B;H1H17996B; H1H17997B; H1H17998B; H1H17999B; H1H18000B; H1H18001B;H1H18002B; H1H18003B; H1H18004B; H1H18005B; H1H18006B; H1H18007B;H1H18008B; H1H18009B; H1H18010B; H1H18011B; H1H18012B; H1H18013B;H1H18014B; H1H18015B; H1H18016B; H1H18017B; H1H18018B; H1H18019B;H1H18020B; H1H18021B; H1H18022B; H1H18023B; H1H18024B; H1H18025B;H1H18026B; H1H18027B; H1H18028B; H1H18029B; H1H18030B; H1H18031B;H1H18032B; H1H18033B; H1H18034B; H1H18035B; H1H18037B; H1H18038B;H1H18039B; H1H18040B; H1H18041B; H1H18042B; H1H18043B; H1H18044B;H1H18045B; H1H18046B; H1H18047B; H1H18048B; H1H18049B; H1H18051B;H1H18052B; H1H18053B; H1H18054B; H1H18055B; H1H18056B; H1H18057B;H1H18058B; H1H18059B; H1H18060B; H1H18061B; H1H18062B; H1H18063B;H1H18064B; H1H18065B; H1H18066B; H1H18067B; H1H18068B; H1H18069B;H1H18070B; H1H18071B; H1H18072B; H1H18073B; H1H18074B; H1H18075B;H1H18076B; H1H18077B; H1H18078B; H1H18079B; H1H18080B; H1H18081B;H1H18082B; H1H18083B; H1H18084B; H1H18085B; H1H18086B; H1H18087B;H1H18088B; H1H18089B; H1H18090B; H1H18091B; H1H18092B; H1H18093B;H1H18094B; H1H18095B; H1H18096B; H1H18097B; H1H18098B; H1H18099B;H1H18100B; H1H18101B; H1H18102B; H1H18103B; H1H18104B; H1H18105B;H1H18107B; H1H18108B; H1H18109B; H1H18110B; H1H18111B; H1H18112B;H1H18113B; H1H18114B; H1H18115B; H1H18116B; H1H18117B; H1H18118B;H1H18119B; H1H18120B; H1H18121B; H1H18122B; H1H18123B; H1H18124B;H1H18125B; H1H18126B; H1H18127B; H1H18128B; H1H18129B; H1H18130B;H1H18131B; H1H18132B; H1H18133B; H1H18134B; H1H18135B; H1H18136B;H1H18137B; H1H18138B; H1H18139B; H1H18140B; H1H18141B; H1H18142B;H1H18143B; H1H18144B; H1H18145B; H1H18146B; H1H18147B; H1H18148B;H1H18149B; H1H18150B; H1H18151B; H1H18152B; H1H18153B; H1H18154B;H1H18155B; H1H18156B; H1H18157B; H1H18158B; H1H18159B; H1H18160B;H1H18161B; H1H18162B; H1H18163B; H1H18164B; H1H18165B; H1H18166B;H1H18167B; H1H18168B; H1H18169B; H1H18170B; H1H18171B; H1H18172B;H1H18173B; H1H18174B; H1H18175B; H1H18176B; H1H18177B; H1H18178B;H1H18179B; H1H18180B; H1H18181B; H1H18182B; H1H18183B; H1H18184B;H1H18185B; H1H18186B; H1H18187B; H1H18188B; H1H18189B; H1H18190B;H1H18191B; H1H18192B; H1H18193B; H1H18194B; H1H18195B; H1H18196B;H1H18197B; H1H18198B; H1H18199B; H1H18200B; H1H18201B; H1H18202B;H1H18203B; H1H18204B; H1H18205B; H1H18206B; H1H18207B; H1H18208B;H1H18209B; H1H18210B; H1H18211B; H1H18212B; H1H18213B; H1H18214B;H1H18216B; H1H18217B; H1H18218B; H1H18219B; H1H18220B; H1H18221B;H1H18222B; H1H18223B; H1H18224B; H1H18225B; H1H18226B; H1H18227B;H1H18228B; H1H18229B; H1H18230B; H1H18231B; H1H18232B; H1H18233B;H1H18234B; H1H18235B; H1H18236B; H1H18237B; H1H18238B; H1H18239B;H1H18240B; H1H18241B; H1H18242B; H1H18243B; H1H18244B; H1H18245B;H1H18246B; H1H18247B; H1H18248B; H1H18249B; H1H18250B; H1H18251B;H1H18252B; H1H18253B; H1H18254B; H1H18255B; H1H18256B; H1H18257B;H1H18258B; H1H18259B; H1H18261B; H1H18262B; H1H18263B; H1H18264B;H1H18265B; H1H18266B; H1H18267B; H1H18268B; H1H18269B; H1H18270B;H1H18271B; H1H18272B; H1H18274B; H1H18275B; H1H18276B; H1H18277B;H1H18278B; H1H18279B; H1H18280B; H1H18281B; H1H18282B; H1H18283B;H1H18284B; H1H18285B; H1H18286B; H1H18287B; H1H18288B; H1H18289B;H1H18290B; H1H18291B; H1H18292B; H1H18293B; H1H18294B; H1H18295B;H1H18297B; H1H18298B; H1H18299B; H1H18300B; H1H18301B; H1H18302B;H1H18303B; H1H18304B; H1H18305B; H1H18306B; H1H18307B; H1H18308B;H1H18309B; H1H18310B; H1H18311B; H1H18312B; H1H18313B; H1H18314B;H1H18315B; H1H18316B; H1H18317B; H1H18318B; H1H18319B; H1H18320B;H1H18321B; H1H18322B; H1H18323B; H1H18324B; H1H18325B; H1H18326B;H1H18327B; H1H18328B; H1H18329B; H1H18330B; H1H18331B; H1H18332B;H1H18333B; H1H18334B; and H1H18335B.
 19. A vessel or injection devicecomprising the antigen-binding protein of claim
 1. 20. A humanantigen-binding protein that specifically binds to human TMPRSS2,comprising: (a) an immunoglobulin heavy chain variable region comprisingthe amino acid sequence set forth in SEQ ID NO: 2; and (b) animmunoglobulin light chain variable region comprising the amino acidsequence set forth in SEQ ID NO: 4 or
 18. 21. The human antigen-bindingprotein of claim 20, comprising: (a) the immunoglobulin heavy chainvariable region comprising the amino acid sequence set forth in SEQ IDNO: 2; and (b) the immunoglobulin light chain variable region comprisingthe amino acid sequence set forth in SEQ ID NO:
 4. 22. The humanantigen-binding protein of claim 21 which is an antibody orantigen-binding fragment thereof.